Compositions and methods for treatment of cardiac diseases

ABSTRACT

Disclosed herein include microRNA antagonists, therapeutic compositions that include one or more of such microRNA antagonists, and methods of treating and/or ameliorating cardiac diseases and/or muscular dystrophy disorders with the microRNA antagonists. Also included are combination therapies, wherein a therapeutic composition disclosed herein and an additional therapy agent are provided to a subject having or suspected of having cardiac disease and/or muscular dystrophy disorder. In particular, some embodiments disclosed herein relate to compositions and methods for transiently administering a mixture of microRNA antagonists for promoting cardiomyocyte proliferation and cardiac regeneration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/318,353 filed on Jan. 16, 2019, which is the U.S. National Phaseunder 35 U.S.C. § 371 of International Application No. PCT/US2017/042400filed on Jul. 17, 2017 designating the U.S., which claims the benefit ofpriority to U.S. Provisional Patent Application Ser. No. 62/363,512,filed on Jul. 18, 2016, and U.S. Provisional Patent Application Ser. No.62/419,852, filed on Nov. 9, 2016. The disclosures of the above-relatedapplications are herein expressly incorporated by reference it theirentireties, including any drawings.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

The present application was made in part with government support underGrant No. R41HL134387 and GRANT12233027 awarded by the National Heart,Lung, And Blood Institute of the National Institutes of Health. Thegovernment has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitled“SequenceListing”, created Aug. 11, 2020, which is approximately 60 KBin size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

FIELD

Aspects of the present application relate to the fields of biochemistryand medicine. More particularly, disclosed herein are novel microRNAantagonists, therapeutic compositions that include one or more of suchmicroRNA antagonists, and methods of treating and/or amelioratingcardiac diseases and/or muscular dystrophy disorders with such microRNAantagonists. Also included are combination therapies, wherein atherapeutic composition disclosed herein and an additional therapy agentare provided to a subject having or suspected of having cardiac diseaseand/or muscular dystrophy disorder where cardiac muscle regeneration isrequired.

BACKGROUND

Heart diseases encompass a family of disorders, including, but notlimited to cardiomyopathies, myocardial infarction, and ischemic heartdisease where cardiac muscle regeneration is required. Ischemic heartdisease is a leading cause of morbidity and mortality in theindustrialized world. Disorders within the heart disease spectrum areunderstood to arise from pathogenic changes in distinct cell types, suchas cardiomyocytes, via alterations in a complex set of biochemicalpathways. For example, certain pathological changes linked with heartdisease can be accounted for by alterations in cardiomyocyte geneexpression that lead to cardiomyocyte hypertrophy and impairedcardiomyocyte survival and contraction. Thus, an ongoing challenge inthe development of heart disease treatments has been to identifyeffective therapies suitable for various types of heart diseases by, forexample, promoting endogenous cardiac myocytes within the heart todivide and repair the damaged cardiac muscle.

The muscular dystrophies (MD) are a group of more than 30 geneticdiseases characterized by progressive weakness and degeneration of theskeletal muscles that control movement. Some forms of MD are seen ininfancy or childhood, while others may not appear until middle age orlater. The disorders differ in terms of the distribution and extent ofmuscle weakness (some forms of MD also affect cardiac muscle), age ofonset, rate of progression, and pattern of inheritance.

In particular, Duchenne muscular dystrophy (DMD) is one of the mostprevalent inherited neuromuscular disorders. Caused by mutations in thedystrophin gene, DMD is characterized by progressive muscle weakness andwasting due to the absence of dystrophin protein resulting indegeneration of skeletal and cardiac muscle with subsequent fibrosis.The common cause of death for people with DMD is cardiomyopathy andheart failure. With no treatment currently available, there is a needfor safe and effective therapies that prevent muscle degeneration inpatients with DMD. The failure of human adult muscle cells to regeneratethemselves constitutes a major clinical problem in DMD. This iscompounded by the lack of adjunctive treatments, pharmacologic orcellular, that can be administered to successfully stimulateregeneration of cardiac muscle. Currently, there is no cure for DMD tofully restore dystrophin protein. With patients having a poor prognosisresulting in premature death, a significant unmet medical need existsfor developing new treatment approaches.

SUMMARY

This section provides a general summary of the disclosure, and is notcomprehensive of its full scope or all of its features.

The present disclosure generally relates to compositions and methods forthe treatment of cardiac diseases and/or muscular dystrophy disorders.Some embodiments of the disclosure relate to the design of therapeuticsand delivery systems of antagonists that specifically target a number ofmicroRNAs of interest, including miR-9a-5p, miR-100-5p, Let-7a-5p,Let-7c-5p. In some embodiments, the compositions and methods disclosedherein allow for regeneration of cardiac muscles and for the treatmentof heart diseases such as, for example, myocardial infarction or anycardiac injury where cardiac muscle regeneration is required. Withoutbeing bound by any particular theory, it is believed that regenerationof damaged cardiac myocytes can potentially lead to a reverse ofischemic injury of heart muscle after a heart attack.

In one aspect, disclosed herein are embodiments of compositions thatinclude a plurality of microRNA (miR) antagonists, wherein the pluralityof miR antagonists includes one or more miR-99a antagonists, one or moremiR-100-5p antagonists, one or more miR-Let-7a-5p antagonists, and oneor more miR-Let-7c-5p antagonists. Implementations of embodiments of thecompositions according to this aspect and other aspects of thedisclosure can include one or more of the following features.

In some embodiments, at least one of the one or more miR-99a antagonistsincludes an anti-miR-99a comprising a nucleotide sequence having atleast 80%, 85%, 90%, 95%, 96%, 97, 98%, 99% or 100% sequence identity toa nucleotide sequence selected from the group consisting of SEQ ID NOs47, 48, 50, 52, and 54. In some embodiments, at least one of the one ormore miR-100-5p antagonists includes an anti-miR-100-5p comprising anucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97, 98%,99% or 100% sequence identity to a nucleotide sequence selected from thegroup consisting of SEQ ID NOs 46, 49, 51, 53, and 55. In someembodiments, at least one of the one or more Let-7a-5p antagonistsincludes an anti-miR-Let-7a-5p comprising a nucleotide sequence havingat least 80%, 85%, 90%, 95%, 96%, 97, 98%, 99% or 100% sequence identityto a nucleotide sequence selected from the group consisting of SEQ IDNOs: 37, 39, and 40-45. In some embodiments, at least one of the one ormore Let-7c-5p antagonists includes an anti-miR-Let-7c-5p comprising anucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97, 98%,99% or 100% sequence identity to a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 36, 38, and 40-45.

In some embodiments, at least one of the one or more miR-99a antagonistsincludes an anti-miR-99a comprising a nucleotide sequence having one ormore mismatched nucleobases with respect to a sequence selected from thegroup consisting of SEQ ID NOs: 47, 48, 50, 52, and 54. In someembodiments, at least one of the one or more miR-100-5p antagonistsincludes an anti-miR-100-5p comprising a nucleotide sequence having oneor more mismatched nucleobases with respect to a sequence selected fromthe group consisting of SEQ ID NOs: 46, 49, 51, 53, and 55. In someembodiments, at least one of the one or more Let-7a-5p antagonistsincludes an anti-miR-Let-7a-5p comprising a nucleotide sequence havingone or more mismatched nucleobases with respect to a sequence selectedfrom the group consisting of SEQ ID NOs: 37, 39, and 40-45. In someembodiments, at least one of the one or more Let-7c-5p antagonistsincludes an anti-miR-Let-7c-5p comprising a nucleotide sequence havingone or more mismatched nucleobases with respect to a sequence selectedfrom the group consisting of SEQ ID NOs: 36, 38, and 40-45.

In various embodiments of the compositions disclosed herein, at leastone of the anti-miRs includes one or more chemical modificationsselected from the group consisting of a modified internucleosidelinkage, a modified nucleotide, and a modified sugar moiety, andcombinations thereof. In some embodiments, the one or more chemicalmodifications includes a modified internucleoside linkage. In someembodiments, the modified internucleoside linkage is selected from thegroup consisting of a phosphorothioate, 2′-Omethoxyethyl (MOE),2′-fluoro, alkylphosphonate, phosphorodithioate, alkylphosphonothioate,phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate,carboxymethyl ester, and combinations thereof. In some embodiments, themodified internucleoside linkage includes a phosphorothioateinternucleoside linkage. In some embodiments, at least one of the one ormore chemical modifications includes a modified nucleotide. In someembodiments, the modified nucleotide includes a locked nucleic acid(LNA) chemistry modification, a peptide nucleic acid (PNA), anarabino-nucleic acid (FANA), an analogue, a derivative, or a combinationthereof. In some embodiments, the modified nucleotide includes a lockednucleic acid (LNA). In some embodiments, the locked nucleic acid (LNA)is incorporated at one or both ends of the modified anti-miR. In someembodiments, at least one of the one or more chemical modificationsincludes a modified sugar moiety. In some embodiments, the modifiedsugar moiety is a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxymodified sugar moiety, a 2′-O-alkyl modified sugar moiety, a bicyclicsugar moiety, or a combination thereof. In some embodiments, themodified sugar moiety comprises a 2′-O-methyl sugar moiety. In someembodiments of the compositions disclosed herein, the composition isfurther formulated into a pharmaceutical formulation.

In one aspect, disclosed herein are embodiments of expression cassettesthat include a nucleotide sequence encoding one or more miR-99aantagonists, one or more miR-100-5p antagonists, one or moremiR-Let-7a-5p antagonists, and one or more miR-Let-7c-5p antagonists. Insome embodiments, at least one of the one or more miR-99a antagonistsincludes an anti-miR-99a comprising a nucleotide sequence having atleast 80%, 85%, 90%, 95%, 96%, 97, 98%, 99% or 100% sequence identity toa nucleotide sequence selected from the group consisting of SEQ ID NOs47, 48, 50, 52, and 54. In some embodiments, at least one of the one ormore miR-100-5p antagonists includes an anti-miR-100-5p comprising anucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97, 98%,99% or 100% sequence identity to a nucleotide sequence selected from thegroup consisting of SEQ ID NOs 46, 49, 51, 53, and 55. In someembodiments, at least one of the one or more Let-7a-5p antagonistsincludes an anti-miR-Let-7a-5p comprising a nucleotide sequence havingat least 80%, 85%, 90%, 95%, 96%, 97, 98%, 99% or 100% sequence identityto a nucleotide sequence selected from the group consisting SEQ ID NOs:37, 39, and 40-45. In some embodiments, at least one of the one or moreLet-7c-5p antagonists includes an anti-miR-Let-7c-5p comprising anucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97, 98%,99% or 100% sequence identity to a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 36, 38, and 40-45.

In various embodiments of the expression cassettes disclosed herein, oneor more of the following applies. In some embodiments, at least one ofthe one or more miR-99a antagonists includes an anti-miR-99a comprisinga nucleotide sequence having one or more mismatched nucleobases withrespect to a sequence selected from the group consisting of SEQ ID NOs:47, 48, 50, 52, and 54. In some embodiments, at least one of the one ormore miR-100-5p antagonists includes an anti-miR-100-5p comprising anucleotide sequence having one or more mismatched nucleobases withrespect to a sequence selected from the group consisting of SEQ ID NOs:46, 49, 51, 53, and 55. In some embodiments, at least one of the one ormore Let-7a-5p antagonists includes an anti-miR-Let-7a-5p comprising anucleotide sequence having one or more mismatched nucleobases withrespect to a sequence selected from the group consisting of SEQ ID NOs:37, 39, and 40-45. In some embodiments, at least one of the one or moreLet-7c-5p antagonists includes an anti-miR-Let-7c-5p comprising anucleotide sequence having one or more mismatched nucleobases withrespect to a sequence selected from the group consisting SEQ ID NOs: 36,38, and 40-45.

In various embodiments of the expression cassettes disclosed herein, oneor more of the following applies. In some embodiments, at least one ofthe anti-miRs includes one or more chemical modifications selected fromthe group consisting of a modified internucleoside linkage, a modifiednucleotide, and a modified sugar moiety, and combinations thereof. Insome embodiments, the one or more chemical modifications includes amodified internucleoside linkage. In some embodiments, the modifiedinternucleoside linkage is selected from the group consisting of aphosphorothioate, 2′-Omethoxyethyl (MOE), 2′-fluoro, alkylphosphonate,phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate,carbonate, phosphate triester, acetamidate, carboxymethyl ester, andcombinations thereof. In some embodiments, the modified internucleosidelinkage includes a phosphorothioate internucleoside linkage. In someembodiments, at least one of the one or more chemical modificationsincludes a modified nucleotide. In some embodiments, the modifiednucleotide includes a locked nucleic acid (LNA) chemistry modification,a peptide nucleic acid (PNA), an arabino-nucleic acid (FANA), ananalogue, a derivative, or a combination thereof. In some embodiments,the modified nucleotide includes a locked nucleic acid (LNA). In someembodiments, the locked nucleic acid (LNA) is incorporated at one orboth ends of the modified anti-miR. In some embodiments, at least one ofthe one or more chemical modifications includes a modified sugar moiety.In some embodiments, the modified sugar moiety is a 2′-O-methoxyethylmodified sugar moiety, a 2′-methoxy modified sugar moiety, a 2′-O-alkylmodified sugar moiety, a bicyclic sugar moiety, or a combinationthereof. In some embodiments, the modified sugar moiety comprises a2′-O-methyl sugar moiety. In some embodiments, the composition accordingto this aspect is a pharmaceutical composition.

In one aspect, some embodiments of the present application relate to acloning vector or expression vector that include an expression cassetteas disclosed herein. In some embodiments, the cloning vector orexpression vector disclosed herein includes an expression cassetteincluding a nucleotide sequence which encodes one or more miR-99aantagonists, one or more miR-100-5p antagonists, one or moremiR-Let-7a-5p antagonists, and one or more miR-Let-7c-5p antagonists. Insome embodiments, the cloning vector or expression vector is a viralvector. In some embodiments, the viral vector is a lentiviral vector oran adeno-associated viral (AAV) vector. In some embodiments, the cloningvector or expression vector disclosed herein includes a nucleotidesequence having at least 80%, 85%, 90%, 95%, 96%, 97, 98%, 99% or 100%sequence identity to each of the nucleotide sequences set forth in SEQID NOs: 59-64; or a nucleotide sequence having at least 80%, 85%, 90%,95%, 96%, 97, 98%, 99% or 100% sequence identity to each of thenucleotide sequences set forth in SEQ ID NOs: 86-89; or a nucleotidesequence having at least 80%, 85%, 90%, 95%, 96%, 97, 98%, 99% or 100%sequence identity to each of the nucleotide sequences set forth in theSEQ ID NOs indicated in a) and b). In some embodiments, the cloningvector or expression vector disclosed herein includes an expressioncassette including a nucleotide sequence having least 80%, 85%, 90%,95%, 96%, 97, 98%, 99% or 100% sequence identity to the nucleotidesequence of SEQ ID NO: 85.

In one aspect, disclosed herein are embodiments of a therapeuticcomposition that includes an effective amount of at least onetherapeutic agent, and one or more of the followings: (a) a compositioncomprising a plurality of microRNA (miR) antagonists as disclosedherein; (b) an expression cassette as disclosed herein; and (c) acloning or expression vector as disclosed herein. In some embodiments,the therapeutic composition is further formulated into a pharmaceuticalformulation.

In some embodiments, the at least one therapeutic agent is selected fromthe group consisting of Idebenone, Eplerenone, VECTTOR, AVI-4658,Ataluren/PTC124/Translarna, BMN044/PRO044, CAT-1004, MicroDystrophin AAVGene Therapy (SGT-001), Galectin-1 Therapy (SB-002), LTBB4 (SB-001),rAAV2.5-CMV-minidystrophin, Glutamine, NFKB inhibitors, Sarcoglycan,delta (35 kDa dystrophin-associated glycoprotein), Insulin like growthfactor-1 (IGF-1), and combinations thereof. In some embodiments, thetherapeutic composition according to this aspect is a pharmaceuticalcomposition.

In one aspect, some embodiments of the disclosure relate to a method fortreating a cardiac disease in a subject. The method includesadministering or providing to the subject a therapeutic compositionsuitable for the treatment of cardiac diseases, wherein (a) thetherapeutic composition is a composition comprising a plurality ofmicroRNA (miR) antagonists as disclosed herein; (b) the therapeuticcomposition comprises an expression cassette as disclosed herein; or (c)the therapeutic composition comprises a cloning or expression vector asdisclosed herein. In some embodiments, the method further includesidentifying the subject as having or suspected of having a cardiacdisease. In some embodiments, the cardiac disease is myocardialinfarction, ischemic heart disease, dilated cardiomyopathy, heartfailure (e.g., congestive heart failure), ischemic cardiomyopathy,hypertrophic cardiomyopathy, restrictive cardiomyopathy, alcoholiccardiomyopathy, viral cardiomyopathy, tachycardia-mediatedcardiomyopathy, stress-induced cardiomyopathy, amyloid cardiomyopathy,arrhythmogenic right ventricular dysplasia, left ventricularnoncompaction, endocardial fibroelastosis, aortic stenosis, aorticregurgitation, mitral stenosis, mitral regurgitation, mitral prolapse,pulmonary stenosis, pulmonary stenosis, pulmonary regurgitation,tricuspid stenosis, tricuspid regurgitation, congenital disorder,genetic disorder, or a combination thereof.

In another aspect, some embodiments of the disclosure relate to a methodfor promoting cardiac muscle regeneration in a subject. The methodincludes administering or providing to the subject a therapeuticcomposition, wherein (a) the therapeutic composition is a compositioncomprising a plurality of microRNA (miR) antagonists as disclosedherein; (b) the therapeutic composition comprises an expression cassetteas disclosed herein; or (c) the therapeutic composition comprises acloning or expression vector as disclosed herein. In some embodiments,the method further includes identifying or selecting the subject ashaving or suspected of having a cardiac disease. In some embodiments,the cardiac disease is myocardial infarction, ischemic heart disease,heart failure (e.g., congestive heart failure), ischemic cardiomyopathy,hypertrophic cardiomyopathy, restrictive cardiomyopathy, alcoholiccardiomyopathy, viral cardiomyopathy, tachycardia-mediatedcardiomyopathy, stress-induced cardiomyopathy, amyloid cardiomyopathy,arrhythmogenic right ventricular dysplasia, left ventricularnoncompaction, endocardial fibroelastosis, aortic stenosis, aorticregurgitation, mitral stenosis, mitral regurgitation, mitral prolapse,pulmonary stenosis, pulmonary stenosis, pulmonary regurgitation,tricuspid stenosis, tricuspid regurgitation, congenital disorder,genetic disorder, or a combination thereof. In some other particularembodiments, the cardiac disease is Ischemic heart disease where cardiacmuscle regeneration is required.

In yet another aspect, some embodiments disclosed herein relate to amethod of modulating proliferation of a cardiomyocyte and/or musclecell. The method includes (1) introducing into a cardiomyocyte atherapeutic composition, wherein (a) the therapeutic composition is acomposition comprising a plurality of microRNA (miR) antagonists asdisclosed herein; (b) the therapeutic composition comprises anexpression cassette as disclosed herein; or (c) the therapeuticcomposition comprises a cloning or expression vector as disclosedherein; and (2) allowing the cardiomyocyte obtained from (1) to divide,thereby modulating proliferation of the cardiomyocyte or muscle cell. Insome embodiments, the introduction of the therapeutic composition intothe cardiomyocyte includes transfecting the cardiomyocyte and/or musclecell with at least one expression cassette or at least one viral vectorcomprising a nucleic acid sequence encoding the plurality of miRantagonists. In some embodiments, the method further includes measuringthe proliferation of the cardiomyocyte and/or muscle cell. In someembodiments, the proliferation of the cardiomyocyte and/or muscle cellis increased compared to a control cardiomyocyte lacking the nucleicacid sequence encoding the plurality of miR antagonists. In someembodiments, the cardiomyocyte and/or muscle is in vivo. In some otherembodiments, the cardiomyocyte and/or muscle is ex vivo. In someembodiments, the cardiomyocyte and/or muscle is of a human subject. Insome embodiments, the human subject is suffering from a cardiac disease.

Implementations of embodiments of the methods disclosed herein caninclude one or more of the following features. In some embodiments, theplurality of miR antagonists are encoded by the same expression cassetteor vector. In some embodiments, the plurality of miR antagonists areencoded by different expression cassettes or vectors. In someembodiments, the vector is a viral vector. In some embodiments, theviral vector is a lentiviral vector or an adeno-associated viral (AAV)vector. In some embodiments, the viral vector is an adeno-associatedviral (AAV) vector.

In some embodiments, the methods further include administrating aneffective amount of at least one additional therapeutic agent or atleast one additional therapy to the subject for a combination therapy.In some embodiments, the at least one additional therapeutic agent ortherapeutic therapy is selected from the group consisting of Idebenone,Eplerenone, VECTTOR, AVI-4658, Ataluren/PTC124/Translarna,BMN044/PRO044, CAT-1004, microDystrophin AAV gene therapy (SGT-001),Galectin-1 therapy (SB-002), LTBB4 (SB-001), rAAV2.5-CMV-minidystrophin,glutamine, NFKB inhibitors, sarcoglycan, delta (35 kDadystrophin-associated glycoprotein), insulin like growth factor-1(IGF-1) expression, genome editing through the CRISPR/Cas9 system, anygene delivery therapy aimed at reintroducing a functional recombinantversion of the dystrophin gene, Exon skipping therapeutics, read-throughstrategies for nonsense mutations, cell-based therapies, utrophinupregulation, myostatin inhibition, anti-inflammatories/anti-oxidants,mechanical support devices, any standard therapy for muscular dystrophy,and combinations thereof. In some embodiments, the at least oneadditional therapeutic agent or therapy comprises a biologic drug. Insome embodiments, the at least one additional therapeutic agent ortherapy comprises a gene therapy or therapeutic gene modulation agent.

In some embodiments, each of the therapeutic composition and the atleast one additional therapeutic agent or therapy is administered in aseparate formulation. In some embodiments, the therapeutic compositionand the at least one additional therapeutic agent or therapy areadministered sequentially. In some embodiments, the therapeuticcomposition and the at least one additional therapeutic agent or therapyare administered concomitantly. In some embodiments, the therapeuticcomposition and the at least one additional therapeutic agent or therapyare administered in rotation. In some the therapeutic composition andthe at least one additional therapeutic agent or therapy areadministered together in a single formulation.

In one aspect, disclosed herein are embodiments of methods for treatinga muscular dystrophy (MD) disorder. The method includes administering orproviding to the subject a therapeutic composition, wherein (a) thetherapeutic composition is a composition comprising a plurality ofmicroRNA (miR) antagonists as disclosed herein; (b) the therapeuticcomposition comprises an expression cassette as disclosed herein; or (c)the therapeutic composition comprises a cloning or expression vector asdisclosed herein, and wherein the administration of the therapeuticcomposition is performed in combination with an effective amount of atleast one additional therapeutic agent or at least one additionaltherapy to provide a combination therapy. In some embodiments, themuscular dystrophy disorder is associated with Amyotrophic LateralSclerosis (ALS), Charcot-Marie-Tooth Disease (CMT), Congenital MuscularDystrophy (CMD), Duchenne Muscular Dystrophy (DMD), Emery-DreifussMuscular Dystrophy (EDMD), Inherited and Endocrine Myopathies, MetabolicDiseases of Muscle, Mitochondrial Myopathies (MM), Myotonic MuscularDystrophy (MMD), Spinal-Bulbar Muscular Atrophy (SBMA), or a combinationthereof.

Also disclosed herein are embodiments of methods for increasingproliferation of a heart cell and/or increasing the expression and/oractivity of proteins involved in muscle structure and/or function and/orregeneration, comprising contacting or providing the heart cell with acombination of (1) a therapeutic composition, wherein (a) thetherapeutic composition is a composition comprising a plurality ofmicroRNA (miR) antagonists as disclosed herein; (b) the therapeuticcomposition comprises an expression cassette as disclosed herein; or (c)the therapeutic composition comprises a cloning or expression vector asdisclosed herein; and (2) at least one additional therapeutic agent ortherapy. In some embodiments, the heart cell is selected from the groupconsisting of cardiac fibroblasts, cardiac myocytes, endothelial cells,and vascular smooth muscle cells (VSMCs). In some embodiments, the heartcell is selected from the group consisting of cardiomyocytes andskeletal muscle cells.

Also disclosed herein are embodiments of methods for inhibiting orreducing expression of a target microRNA (miR), comprising contacting orproviding a heart cell with a combination of (1) a therapeuticcomposition, wherein (a) the therapeutic composition is a compositioncomprising a plurality of microRNA (miR) antagonists as disclosedherein; (b) the therapeutic composition comprises an expression cassetteas disclosed herein; or (c) the therapeutic composition comprises acloning or expression vector as disclosed herein; and (2) at least oneadditional therapeutic agent or therapy. In some embodiments, the heartcell is selected from the group consisting of cardiac fibroblasts,cardiac myocytes, endothelial cells, and vascular smooth muscle cells(VSMCs). In some embodiments, the heart cell is selected from the groupconsisting of cardiomyocytes and skeletal muscle cells.

Implementations of embodiments of the methods according to the foregoingaspects of the disclosure can include one or more of the followingfeatures. In some embodiments, the at least one additional therapeuticagent or therapeutic therapy is selected from the group consisting ofIdebenone, Eplerenone, VECTTOR, AVI-4658, Ataluren/PTC124/Translarna,BMN044/PRO044, CAT-1004, microDystrophin AAV gene therapy (SGT-001),Galectin-1 therapy (SB-002), LTBB4 (SB-001), rAAV2.5-CMV-minidystrophin,glutamine, NFKB inhibitors, sarcoglycan, delta (35 kDadystrophin-associated glycoprotein), insulin like growth factor-1(IGF-1) expression modulation, genome editing through the CRISPR/Cas9system, any gene delivery therapy aimed at reintroducing a functionalrecombinant version of the dystrophin gene, Exon skipping therapeutics,read-through strategies for nonsense mutations, cell-based therapies,utrophin upregulation, myostatin inhibition,anti-inflammatories/anti-oxidants, mechanical support devices, anystandard therapy for muscular dystrophy, and combinations thereof. Insome embodiments, the at least one additional therapeutic agent ortherapy includes a biologic drug. In some embodiments, the at least oneadditional therapeutic agent or therapy comprises a gene therapy ortherapeutic gene modulation agent. In some embodiments, each of thetherapeutic composition and the at least one additional therapeuticagent or therapy is administered in a separate formulation. In someembodiments, the therapeutic composition and the at least one additionaltherapeutic agent or therapy are administered sequentially. In someembodiments, the therapeutic composition and the at least one additionaltherapeutic agent or therapy are administered concomitantly. In someembodiments, the therapeutic composition and the at least one additionaltherapeutic agent or therapy are administered in rotation. In someembodiments, the therapeutic composition and the at least one additionaltherapeutic agent or therapy are administered in a single formulation.

Disclosed herein further includes microRNA (miR) antagonists. In someembodiments, the miR antagonist include (a) a nucleotide sequence havingat least 80%, 85%, 90%, 95%, 96%, 97, 98%, 99% or 100% sequence identityto a nucleotide sequence selected from the group consisting of SEQ IDNOs 47, 48, 50, 52, and 54; (b) a nucleotide sequence having at least80%, 85%, 90%, 95%, 96%, 97, 98%, 99% or 100% sequence identity to anucleotide selected from the group consisting of SEQ ID NOs 46, 49, 51,53, and 55; or (c) a nucleotide sequence having at least 80%, 85%, 90%,95%, 96%, 97, 98%, 99% or 100% sequence identity to a nucleotideselected from the group consisting of SEQ ID NOs: 37, 39, and 40-45; or(d) a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97,98%, 99% or 100% sequence identity to a nucleotide selected from thegroup consisting of SEQ ID NOs: 36, 38, and 40-45.

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative embodiments andfeatures described herein, further aspects, embodiments, objects andfeatures of the disclosure will become fully apparent from the drawingsand the detailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a non-limiting exemplary cloningvector design which includes nucleotide sequences encoding a modifiedhairpin Zip construct expressing Let-7a-5p and miR-99a-5p inhibitorysequences under control of the H1 promoter and U6 promoter,respectively. In this exemplary illustration, the vector also includesnucleotide sequences encoding a Let-7c-5p and miR-100-5p inhibitorysequences under the regulation of the H1 and U6 promoter, respectively.

FIGS. 2A-2B pictorially summarize the results of cardiac MRI imagingexperiments in which the cardiac MRI images of control GFP virus (FIG.2A) versus JBT-miR1 (FIG. 2B) were observed to decrease late gadoliniumenhancement of the Left Ventricle (LV) in CD1 mice with permanent LADligation 3 weeks following an intracardiac injection of JBT-miR1 whencompared with a virus expressing GFP.

FIG. 3 is a schematic representation of a non-limiting exemplarypMIR-REPORT™ Luciferase miRNA expression reporter vector that contains afirefly luciferase reporter gene.

FIG. 4 is a schematic representation of a non-limiting exemplarypMIR-REPORT™ miRNA β-Galactosidase expression reporter vector thatcontains a β-Galactosidase reporter gene.

FIG. 5 is a schematic summary of the results of experiments performed inHela cells, demonstrating that the endogenous miRs (Let-7a-5p miR-99a,miR-100-5p, miR-Let-7c5p, miR-Let-7a-5p) within Hela cells can bind tothe respective LUC reporter constructs described in Example 5 below, andrepress luciferase activity.

FIG. 6 is a schematic summary of the results of experiments performed inHela cells, illustrating that JRX0111, JRX0112, JRX0114, JRX0116,JRX0118 miR-99a (miR-99) anti-miRs were found to increase LuciferaseConstruct 1 (LUC 1, miR-99a) activity in a dose-dependent manner (Log-10M) which contained a miR binding sequence complementary to miR-99acloned into the multiple cloning site of pMIR-REPORT™ Luciferase (pMIR).

FIG. 7 is a schematic summary of the results of experiments performed inHela cells, demonstrating that JRX0110, JRX0113, JRX0115, JRX0117,JRX0119 miR-100-5p anti-miRs were observed to increase LuciferaseConstruct 2 (LUC 2, miR-100-5p) activity in a dose-dependent manner(Log-10 M) which contained a miR binding sequence complementary tomiR-100-5p cloned into the multiple cloning site of pMIR-REPORT™Luciferase (pMIR).

FIGS. 8A-8B schematically summarize the results of experiments performedin Hela cells, demonstrating that JRX0101, JRX0103, JRX0104, JRX0105,JRX0106, JRX0107, JRX0108, JRX0109 Let-7a-5p miR-Let-7a-5p anti-miRswere found to increase Luciferase Construct 3 (LUC 3, let-7a) activityin a dose-dependent manner (Log-10 M). LUC 3 contained a miR bindingsequence complementary to miR-Let-7a-5p cloned into the multiple cloningsite of pMIR-REPORT™ Luciferase (pMIR).

FIGS. 9A-9B schematically summarize of the results of experimentsperformed in Hela cells, demonstrating that JRX0100, JRX0102, JRX0104,JRX0105, JRX0106, JRX0107, JRX0108, JRX0109 Let-7c-5p miR-Let-7c5panti-miRs were observed to increase Luciferase Construct 4 (LUC 4,let-7c) activity in a dose-dependent manner (Log-10 M). LUC 4 containeda miR binding sequence complementary to miR-Let-7c5p cloned into themultiple cloning site of pMIR-REPORT™ Luciferase (pMIR).

FIG. 10 is a schematic summary of the results of experiments performedin neonatal rat ventricular cardiac myocytes, demonstrating thatexperimental results demonstrating that JRX0111, JRX0112, JRX0114,JRX0116, JRX0118 miR-99a anti-miRs were observed to increase LuciferaseConstruct 1 (LUC 1, miR-99) activity in a dose-dependent manner (Log-10M).

FIG. 11 is a schematic summary of the results of experiments performedin neonatal rat ventricular cardiac myocytes, demonstrating thatJRX0110, JRX0113, JRX0115, JRX0117, JRX0119 miR-100-5p anti-miRs wereobserved to increase Luciferase Construct 2 (LUC 2, miR-100) activity ina dose-dependent manner (Log-10 M).

FIGS. 12A-12B schematically summarize the results of experimentsperformed in neonatal rat ventricular cardiac myocytes, demonstratingthat JRX0101, JRX0103, JRX0104, JRX0105, JRX0106, JRX0107, JRX0108,JRX0109 Let-7a-5p miR-Let-7a-5p anti-miRs were found to increaseLuciferase Construct 3 (LUC 3, let-7a) activity in a dose-dependentmanner (Log-10 M).

FIGS. 13A-13B schematically summarize the results of experimentsperformed in neonatal rat ventricular cardiac myocytes, demonstratingthat JRX0100, JRX0102, JRX0104, JRX0105, JRX0106, JRX0107, JRX0108,JRX0109 Let-7c-5p miR-Let-7c5p anti-miRs were observed to increaseLuciferase Construct 4 (LUC 4, let-7c) activity in a dose-dependentmanner (Log-10 M).

The foregoing and other features of the present disclosure will becomemore fully apparent from the following description and appended claims,taken in conjunction with the accompanying drawings. Understanding thatthese drawings depict only several embodiments in accordance with thedisclosure and are not to be considered limiting of its scope; thedisclosure will be described with additional specificity and detailthrough use of the accompanying drawings.

DETAILED DESCRIPTION

The present disclosure generally relates to novel microRNA antagonists,therapeutic compositions that include one or more of such microRNAantagonists, and methods of treating and/or ameliorating cardiacdiseases and/or muscular dystrophy disorders with such microRNAantagonists. Also included are combination therapies wherein atherapeutic composition disclosed herein and an additional therapy agentare provided to a subject having or suspected of having cardiac diseaseand/or muscular dystrophy disorder. In particular, some embodimentsdisclosed herein relate to the use of various combinations of syntheticoligonucleotide miR-99A-5P, miR-100-5P, Let-7a-5p, and Let-7c-5pantagonists and/or viral delivered miR-99A-5P, miR-100-5P, Let-7a-5p,and Let-7c-5p antagonists, chemotherapeutic agents, and biologicalagents for the treatment of cardiac diseases and/or muscular dystrophydisorders. For example, some embodiments disclosed herein describe twoadenoviral AAV2/9 delivery systems (referred to herein as JBT-miR1 andJBT-miR2), and the corresponding expression vectors with a number ofvariants for miR-99a-5p, miR-100-5p, Let-7a-5p, Let-7c-5p antagoniststhat are capable of inhibiting the respective target microRNAs. Furtherprovided herein are a number of synthetic oligonucleotide antagonistsdesigned for specifically targeting miR-99a-5p, miR-100-5p, Let-7a-5p,and Let-7c, individually or in combination.

In the following detailed description, reference is made to theaccompanying Figures, which form a part hereof. The illustrativeembodiments described in the detailed description, Figures, and claimsare not meant to be limiting. Other embodiments may be used, and otherchanges may be made, without departing from the spirit or scope of thesubject matter presented here. It will be readily understood that theembodiments of the present disclosure, as generally described herein,and illustrated in the Figures, can be arranged, substituted, combined,and designed in a wide variety of different configurations, all of whichare explicitly contemplated and make part of this disclosure.

Some Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisdisclosure pertains when read in light of this disclosure. In somecases, terms with commonly understood meanings are defined herein forclarity and/or for ready reference, and the inclusion of suchdefinitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art.Many of the techniques and procedures described or referenced herein arewell understood and commonly employed using conventional methodology bythose skilled in the art. (See, e.g., Singleton et al., Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual,Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989).

The singular form “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise. For example, the term “amolecule” includes one or more molecules, including mixtures thereof. Asused in this disclosure and the appended claims, the term “and/or” canbe singular or inclusive. For example, “A and/or B” is used herein toinclude all of the following alternatives: “A”, “B”, and “A and B”.

The term “about”, as used herein, has its ordinary meaning ofapproximately. If the degree of approximation is not otherwise clearfrom the context, “about” means either within plus or minus 10% of theprovided value, or rounded to the nearest significant figure, in allcases inclusive of the provided value. Where ranges are provided, theyare inclusive of the boundary values.

“Administering” means providing a pharmaceutical agent or composition toa subject, and includes, but is not limited to, administering by amedical professional and self-administering.

“Parenteral administration,” means administration through injection orinfusion. Parenteral administration includes, but is not limited to,subcutaneous administration, intravenous administration, intramuscularadministration, intra-arterial administration, and intracranialadministration. “Subcutaneous administration” means administration justbelow the skin. “Intravenous administration” means administration into avein. “Intraarterial administration” means administration into anartery.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, y-carboxyglutamate, and 0-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, e.g., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. The terms“non-naturally occurring amino acid” and “unnatural amino acid” refer toamino acid analogs, synthetic amino acids, and amino acid mimetics whichare not found in nature.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Antisense compound” means a compound having a nucleobase sequence thatwill allow hybridization to a target nucleic acid. In certainembodiments, an antisense compound is an oligonucleotide having anucleobase sequence complementary to a target nucleic acid.

The terms “complementary” or “complementarity” refer to the ability of anucleic acid in a polynucleotide to form a base pair with anothernucleic acid in a second polynucleotide. For example, the sequence A-G-Tis complementary to the sequence T-C-A. Complementarity may be partial,in which only some of the nucleic acids match according to base pairing,or complete, where all the nucleic acids match according to basepairing. The terms “protein”, “peptide”, and “polypeptide” are usedinterchangeably to denote an amino acid polymer or a set of two or moreinteracting or bound amino acid polymers. These terms, as used herein,encompass amino acid polymers in which one or more amino acid residue isan artificial chemical mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers andnon-naturally occurring amino acid polymer.

The phrase “conservatively modified variants” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical nucleotide sequences. Because of thedegeneracy of the genetic code, a large number of functionally identicalnucleic acids can encode any given protein. For instance, the codonsGCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at everyposition where an alanine is specified by a codon, the codon can bealtered to any of the corresponding codons described above withoutaltering the encoded polypeptide. Such nucleic acid variations are“silent variations,” which are one species of conservatively modifiedvariations. Any one of the nucleic acid sequences described herein whichencodes a polypeptide also describes every possible silent variation ofthe nucleic acid. One of ordinary skill in the art will recognize thateach codon in a nucleic acid (except AUG, which is ordinarily the onlycodon for methionine, and TGG, which is ordinarily the only codon fortryptophan) can be modified to yield a functionally identical molecule.Accordingly, all silent variations of a nucleic acid which encodes apolypeptide are implicit in each of the described sequences with respectto its expression product, but not with respect to actual probesequences. In addition or alternatively, a variant can comprisesdeletions, substitutions, additions of one or more nucleotides at the 5′end, 3′ end, and/or one or more internal sites in comparison to thereference polynucleotide. Similarities and/or differences in sequencesbetween variants and the reference polynucleotide can be detected usingconventional techniques known in the art, for example polymerase chainreaction (PCR) and hybridization techniques. Variant polynucleotidesalso include synthetically derived polynucleotides, such as thosegenerated, for example, by using site-directed mutagenesis. Generally, avariants of a particular polynucleotide disclosed herein, including, butnot limited to, a miRNA, will have at least about 50%, about 55%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99% or more sequence identity to the referencepolynucleotide as determined by sequence alignment programs known byskilled artisan.

The terms “identical” or “percent identity”, in the context of two ormore nucleic acids or proteins, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acids that are the same (e.g., about 60% sequenceidentity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or higher identity over a specified region, when comparedand aligned for maximum correspondence over a comparison window ordesignated region) as measured using a BLAST or BLAST 2.0 sequencecomparison algorithms with default parameters described below, or bymanual alignment and visual inspection. See e.g., the NCBI web site atncbi.nlm.nih.gov/BLAST. Such sequences are then said to be“substantially identical.” This definition also refers to, or may beapplied to, the complement of a test sequence. This definition alsoincludes sequences that have deletions and/or additions, as well asthose that have substitutions. Sequence identity typically exists over aregion that is at least about 50 amino acids or nucleotides in length,or over a region that is 50-100 amino acids or nucleotides in length, orover the entire length of a given sequence.

As used herein, the term “construct” is intended to mean any recombinantnucleic acid molecule such as an expression cassette, plasmid, cosmid,virus, autonomously replicating polynucleotide molecule, phage, orlinear or circular, single-stranded or double-stranded, DNA or RNApolynucleotide molecule, derived from any source, capable of genomicintegration or autonomous replication, comprising a nucleic acidmolecule where one or more nucleic acid sequences has been linked in afunctionally operative manner, e.g. operably linked.

The term “transfection” or “transfecting” is defined as a process ofintroducing a nucleic acid molecule to a cell using non-viral orviral-based methods. The nucleic acid molecule can be a sequenceencoding complete proteins or functional portions thereof. Typically, anucleic acid vector comprises the elements necessary for proteinexpression (e.g., a promoter, transcription start site, etc.). Non-viralmethods of transfection include any appropriate transfection method thatdoes not use viral DNA or viral particles as a delivery system tointroduce the nucleic acid molecule into the cell. Exemplary non-viraltransfection methods include, but are not limited to, calcium phosphatetransfection, liposomal transfection, nucleofection, sonoporation,transfection through heat shock, magnetifection, and electroporation.For viral-based methods, any one of useful viral vectors known in theart can be used in the methods described herein. Examples of viralvectors include, but are not limited to retroviral, adenoviral,lentiviral and adeno-associated viral vectors. In some aspects, thenucleic acid molecules are introduced into a cell using a retroviralvector following standard procedures known in the art.

The term “heterologous” when used with reference to portions of anucleic acid or protein indicates that the nucleic acid or proteincomprises two or more subsequences that are not found in the samerelationship to each other in nature. For instance, the nucleic acid istypically recombinantly produced, having two or more sequences fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source.Similarly, a heterologous protein indicates that the protein comprisestwo or more subsequences that are not found in the same relationship toeach other in nature (e.g., a fusion protein).

The term “gene” is used broadly to refer to any segment of nucleic acidmolecule that encodes a protein or that can be transcribed into afunctional RNA. Genes may include sequences that are transcribed but arenot part of a final, mature, and/or functional RNA transcript, and genesthat encode proteins may further comprise sequences that are transcribedbut not translated, for example, 5′ untranslated regions (5′-UTR), 3′untranslated regions (3′-UTR), introns, etc. Further, genes mayoptionally further comprise regulatory sequences required for theirexpression, and such sequences may be, for example, sequences that arenot transcribed or translated. Genes can be obtained from a variety ofsources, including cloning from a source of interest or synthesizingfrom known or predicted sequence information, and may include sequencesdesigned to have desired parameters.

The term “internucleoside linkage” means a covalent linkage betweenadjacent nucleosides.

The term “nucleobase” means a heterocyclic moiety capable ofnon-covalently pairing with another nucleobase.

“Nucleoside” means a nucleobase linked to a sugar. “Linked nucleosides”means nucleosides joined by a covalent linkage. “Nucleotide” means anucleoside having a phosphate group covalently linked to the sugarportion of a nucleoside.

“miR antagonist” means an agent designed to interfere with or inhibitthe activity of a miRNA. In certain embodiments, a miR antagonistcomprises an antisense compound targeted to a miRNA. In certainembodiments, a miR antagonist comprises a modified oligonucleotidehaving a nucleobase sequence that is complementary to the nucleobasesequence of a miRNA, or a precursor thereof. In certain embodiments, amiR antagonist comprises a small molecule, or the like that interfereswith or inhibits the activity of an miRNA.

“miR-9a-5p antagonist” means an agent designed to interfere with orinhibit the activity of miR-9a-5p. “miR-100-5p antagonist” means anagent designed to interfere with or inhibit the activity of miR-100-5p.“Let-7a-5p antagonist” means an agent designed to interfere with orinhibit the activity of Let-7a-5p. “Let-7c-5p antagonist” means an agentdesigned to interfere with or inhibit the activity of Let-7c-5p.

“Modified oligonucleotide” means an oligonucleotide having one or morechemical modifications relative to a naturally occurring terminus,sugar, nucleobase, and/or internucleoside linkage.

“Modified internucleoside linkage” means any change from a naturallyoccurring internucleoside linkage.

“Phosphorothioate internucleoside linkage” means a linkage betweennucleosides where one of the non-bridging atoms is a sulfur atom.

“Modified sugar” means substitution and/or any change from a naturalsugar.

“Modified nucleobase” means any substitution and/or change from anatural nucleobase.

“5-methylcytosine” means a cytosine modified with a methyl groupattached to the 5′ position.

“2′-O-methyl sugar” or “2′-OMe sugar” means a sugar having an O-methylmodification at the 2′ position.

“2′-O-methoxyethyl sugar” or “2′-MOE sugar” means a sugar having anO-methoxyethyl modification at the 2′ position.

“2′-O-fluoro sugar” or “2′-F sugar” means a sugar having a fluoromodification of the 2′ position.

“Bicyclic sugar moiety” means a sugar modified by the bridging of twonon-geminal ring atoms.

“2′-O-methoxyethyl nucleoside” means a 2′-modified nucleoside having a2′-O-methoxyethyl sugar modification.

“2′-fluoro nucleoside” means a 2′-modified nucleoside having a 2′-fluorosugar modification.

“2′-O-methyl” nucleoside means a 2′-modified nucleoside having a2′-O-methyl sugar modification.

“Bicyclic nucleoside” means a 2′-modified nucleoside having a bicyclicsugar moiety.

As used herein, the terms “miR,” “mir,” and “miRNA” are usedinterchangeably and to refer to microRNA, a class of small RNA moleculesthat are capable of hybridizing to and regulating the expression of acoding RNA. In certain embodiments, a miRNA is the product of cleavageof a pre-miRNA by the enzyme Dicer. These terms as provided herein referto a nucleic acid that forms a double stranded RNA which has the abilityto reduce or inhibit expression of a gene or target gene when expressedin the same cell as the gene or target gene. The complementary portionsof the nucleic acid that hybridize to form the double stranded moleculetypically have substantial or complete identity. In one embodiment, a“microRNA” refers to a nucleic acid that has substantial or completeidentity to a target gene and forms a double stranded miRNA. In someembodiments, the miRNA of the disclosure inhibits gene expression byinteracting with a complementary cellular mRNA thereby interfering withthe expression of the complementary mRNA. In some embodiments, thedouble stranded miRNA of the present disclosure is at least about 15-50nucleotides in length (e.g., each complementary sequence of the doublestranded miRNA is 15-50 nucleotides in length, and the double strandedmiRNA is about 15-50 base pairs in length). In some embodiments, thelength is 20-30 base nucleotides, preferably about 20-25 or about 24-29nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 nucleotides in length. In some embodiments of the disclosure, themicroRNA is selected from, or substantially similar to a microRNAselected from, the group consisting of miR-9a-5p, miR-100-5p, Let-7a-5p,and Let-7c-5p.

As used herein, the term “anti-miRNA” is used interchangeably with theterm “anti-miR”, which refers to an oligonucleotide capable ofinterfering with or inhibiting one or more activities of one or moretarget microRNAs. In some embodiments, the anti-miRNA is a chemicallysynthesized oligonucleotide. In some embodiments, the anti-miRNA is asmall molecule. In some embodiments, the anti-miRNA is a miR antisensemolecule. “Seed region” means nucleotides 2 to 6 or 2 to 7 from the5′-end of a mature miRNA sequence.

The term “miRNA precursor” means a transcript that originates from agenomic DNA and that comprises a non-coding, structured RNA comprisingone or more miRNA sequences. For example, in certain embodiments a miRNAprecursor is a pre-miRNA. In certain embodiments, a miRNA precursor is apri-miRNA.

“Pre-miRNA” or “pre-miR” means a non-coding RNA having a hairpinstructure, which contains a miRNA. In certain embodiments, a pre-miRNAis the product of cleavage of a pri-miR by the double-strandedRNA-specific ribonuclease known as Drosha. Without wishing to be boundby any particular theory, it is believed that in the cytoplasm, thepre-miRNA hairpin is cleaved by the RNase III enzyme Dicer. Thisendoribonuclease interacts with 5′ and 3′ ends of the hairpin and cutsaway the loop joining the 3′ and 5′ arms, yielding an imperfectmiRNA:miRNA duplex of about 22 nucleotides in length. Although eitherstrand of the duplex may potentially act as a functional miRNA, it isbelieved that only one strand is usually incorporated into theRNA-induced silencing complex (RISC) where the miRNA and its mRNA targetinteract. The remaining strand—sense strand—is degraded. The RNA-inducedsilencing complex, or RISC, is a multiprotein complex, specifically aribonucleoprotein, which incorporates one strand of a single-strandedRNA (ssRNA) fragment, such as microRNA (miRNA), or double-stranded smallinterfering RNA (siRNA).

“Modulation” means to a perturbation of function or activity. In certainembodiments, modulation means an increase in gene expression. In certainembodiments, modulation means a decrease in gene expression. The term“microRNA modulator” as used herein refers to an agent capable ofmodulating the level of expression of a microRNA (e.g., let-7 a, let-7c, miR-100, miR-99). In some embodiments, the microRNA modulator isencoded by a nucleic acid. In other embodiments, the microRNA modulatoris a small molecule (e.g., a chemical compound or synthetic microRNAmolecule). In some embodiments, the microRNA modulator decreases thelevel of expression of a microRNA compared to the level of expression inthe absence of the microRNA modulator. Where the microRNA modulatordecreases the level of expression of a microRNA relative to the absenceof the modulator, the microRNA modulator is an antagonist of the microRNA. In some embodiments, the microRNA modulator increases the levelexpression of a micro RNA compared to the level of expression in theabsence of the microRNA modulator. Where the microRNA modulatorincreases the level of expression of a micro RNA relative to the absenceof the modulator, the microRNA modulator is an agonist of the microRNA.

As used herein, the term “myocardial cell” includes any cell that isobtained from, or present in, myocardium such as a human myocardiumand/or any cell that is associated, physically and/or functionally, withmyocardium. In some embodiments disclosed herein, a myocardial cell is acardiomyocyte.

The term “nucleotide” covers naturally occurring nucleotides as well asnon-naturally occurring nucleotides. Thus, “nucleotides” includes notonly the known purine and pyrimidine heterocycles-containing molecules,but also heterocyclic analogues and tautomers thereof. Non-limitingexamples of other types of nucleotides are molecules containing adenine,guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine,8-oxo-N6-methyladenine, 7-deazaxanthine, 7-deazaguanine,N4,N4-ethanocytosin, N6,N6-ethano-2,6-diaminopurine, 5-methylcytosine,5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine,isoguanin, inosine and the “non-naturally occurring” nucleotidesdescribed in U.S. Pat. No. 5,432,272. The term “nucleotide” is intendedto cover every and all of these examples as well as analogues andtautomers thereof.

The term “nucleic acid” and “polynucleotide” are used interchangeablyherein and refer to deoxyribonucleotides or ribonucleotides and polymersthereof in either single- or double-stranded form, and complementsthereof. The term “polynucleotide” include linear sequences ofnucleotides. The term “nucleotide” typically refers to a single unit ofa poly-nucleotide, e.g., a monomer. Nucleotides can be ribonucleotides,deoxyribonucleotides, or modified versions thereof. Examples ofpolynucleotides contemplated herein include single and double strandedDNA, single and double stranded RNA (including siRNA), and hybridmolecules having mixtures of single and double stranded DNA and RNA. Theterms also encompass nucleic acids containing known nucleotide analogsor modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, which have similarbinding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, and2′-O-methyl ribonucleotides. As such, the term “nucleic acid” and“polynucleotide” encompass nucleic acids comprising phosphodiesterlinkages or modified linkages such as phosphotriester, phosphoramidate,siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate,thioether, bridged phosphoramidate, bridged methylene phosphonate,bridged phosphoramidate, bridged phosphoramidate, bridged methylenephosphonate, phosphorothioate, methylphosphonate, phosphorodithioate,bridged phosphorothioate or sultone linkages, and combinations of suchlinkages. The terms “nucleic acid” and “polynucleotide” alsospecifically include nucleic acids composed of bases other than the fivebiologically occurring bases (adenine, guanine, thymine, cytosine anduracil.

The term “operably linked”, as used herein, denotes a functional linkagebetween two or more sequences. For example, an operably linkage betweena polynucleotide of interest and a regulatory sequence (for example, apromoter) is functional link that allows for expression of thepolynucleotide of interest. In this sense, the term “operably linked”refers to the positioning of a regulatory region and a coding sequenceto be transcribed so that the regulatory region is effective forregulating transcription or translation of the coding sequence ofinterest. In some embodiments disclosed herein, the term “operablylinked” denotes a configuration in which a regulatory sequence is placedat an appropriate position relative to a sequence that encodes apolypeptide or functional RNA such that the control sequence directs orregulates the expression or cellular localization of the mRNA encodingthe polypeptide, the polypeptide, and/or the functional RNA. Thus, apromoter is in operable linkage with a nucleic acid sequence if it canmediate transcription of the nucleic acid sequence. Operably linkedelements may be contiguous or non-contiguous.

The terms “promoter”, “promoter region”, or “promoter sequence”, as usedinterchangeably herein, refer to a nucleic acid sequence capable ofbinding RNA polymerase to initiate transcription of a gene in a 5′ to 3′(“downstream”) direction. The specific sequence of the promotertypically determines the strength of the promoter. For example, a strongpromoter leads to a high rate of transcription initiation. A gene is“under the control of” or “regulated by” a promoter when the binding ofRNA polymerase to the promoter is the proximate cause of said gene'stranscription. The promoter or promoter region typically provides arecognition site for RNA polymerase and other factors necessary forproper initiation of transcription. A promoter may be isolated from the5′ untranslated region (5′ UTR) of a genomic copy of a gene.Alternatively, a promoter may be synthetically produced or designed byaltering known DNA elements. Also considered are chimeric promoters thatcombine sequences of one promoter with sequences of another promoter. Apromoter can be used as a regulatory element for modulating expressionof an operably linked polynucleotide molecule such as, for example, acoding sequence of a polypeptide or a functional RNA sequence. Promotersmay contain, in addition to sequences recognized by RNA polymerase and,preferably, other transcription factors, regulatory sequence elementssuch as cis-elements or enhancer domains that affect the transcriptionof operably linked genes. In some embodiments, a promoter can be“constitutive.” In some embodiments, a promoter may be regulated in a“tissue-specific” or “tissue-preferred” manner, such that it is onlyactive in transcribing the operable linked coding region in a specifictissue type or types. In some embodiments, for therapeutic purposes, thepromoter can be a tissue-specific promoter which supports transcriptionin cardiac and skeletal muscle cell. Further information in this regardcan be found in, for example, PCT Patent Publication WO2004041177A2,which is hereby incorporated by reference in its entirety. In someembodiments, a promoter may comprise “naturally-occurring” or“synthetically” assembled nucleic acid sequences.

Expression of a transfected gene can occur transiently or stably in ahost cell. During “transient expression” the transfected nucleic acid isnot integrated into the host cell genome, and is not transferred to thedaughter cell during cell division. Since its expression is restrictedto the transfected cell, expression of the gene can be lost over time.In contrast, stable expression of a transfected gene can occur when thegene is co-transfected with another gene that confers a selectionadvantage to the transfected cell. Such a selection advantage may be aresistance towards a certain toxin that is presented to the cell.Expression of a transfected gene can further be accomplished bytransposon-mediated insertion into to the host genome. Duringtransposon-mediated insertion, the gene is positioned in a predictablemanner between two transposon linker sequences that allow insertion intothe host genome as well as in subsequent excision.

The terms “inhibitor,” “repressor” or “antagonist” or “downregulator”,as used interchangeably herein, refer to a substance, agent, or moleculethat results in a detectably lower expression or activity level of atarget gene as compared to a control. The inhibited expression oractivity can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less thanthat in a control. In some embodiments, the inhibition is 1.5-fold,2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more in comparison to acontrol. In some embodiments, an antagonist is an anti-miR.

As used herein, “treatment” refers to a clinical intervention made inresponse to a disease, disorder or physiological condition manifested bya patient or to which a patient may be susceptible. The aim of treatmentincludes, but is not limited to, the alleviation or prevention ofsymptoms, slowing or stopping the progression or worsening of a disease,disorder, or condition and/or the remission of the disease, disorder orcondition. “Treatments” refer to one or both of therapeutic treatmentand prophylactic or preventative measures. Subjects in need of treatmentinclude those already affected by a disease or disorder or undesiredphysiological condition as well as those in which the disease ordisorder or undesired physiological condition is to be prevented. Insome embodiments of the disclosure, the terms “treatment,” “therapy,”and “amelioration” refer to any reduction in the severity of symptoms,e.g., of a neurodegenerative disorder or neuronal injury. As usedherein, the terms “treat” and “prevent” are not intended to be absoluteterms. Treatment can refer to any delay in onset, amelioration ofsymptoms, and improvement in patient survival, increase in survival timeor rate, etc., or a combination thereof. The effect of treatment can becompared to an individual or pool of individuals not receiving thetreatment, or to the same patient prior to treatment or at a differenttime during treatment. In some embodiments, the severity of disease ordisorder in an individual can be reduced by at least 10%, as compared,e.g., to the individual before administration or to a control individualnot undergoing treatment. In some embodiments, the severity of diseaseor disorder in an individual is reduced by at least 25%, 50%, 75%, 80%,or 90%, or in some embodiments, no longer detectable using standarddiagnostic techniques.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” refers to an amount sufficient to effect beneficial ordesirable biological and/or clinical results. In some embodiments, theterm refers to that amount of the therapeutic agent sufficient toameliorate a given disorder or symptoms. For example, for the givenparameter, a therapeutically effective amount can show an increase ordecrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%,90%, or at least 100% compared to a control. Therapeutic efficacy canalso be expressed as “-fold” increase or decrease. For example, atherapeutically effective amount can have at least a 1.2-fold, 1.5-fold,2-fold, 5-fold, or more effect over a control.

The terms “subject,” “patient,” “individual in need of treatment” andlike terms are used interchangeably and refer to, except whereindicated, an mammal subject that is the object of treatment,observation, or experiment. As used herein, “mammal” refers to a subjectbelonging to the class Mammalia and includes, but not limited to,humans, domestic and farm animals, zoo animals, sports and pet animals.Non-limiting examples of mammals include humans, and non-human primates,mice, rats, sheep, dogs, horses, cats, cows, goats, pigs, and othermammalian species. In some embodiments, the mammal is a human. However,in some embodiments, the mammal is not a human. The term does notnecessarily indicate that the subject has been diagnosed with aparticular disease or disorder, but typically refers to a subject undermedical supervision. “Subject suspected of having” means a subjectexhibiting one or more clinical indicators of a disease or condition. Incertain embodiments, the disease or condition is a muscular dystrophy(MD) disorder.

“Target nucleic acid,” “target RNA,” “target RNA transcript” and“nucleic acid target” all mean a nucleic acid capable of being targetedby antagonists. “Targeting” means the process of design and selection ofnucleobase sequence that will hybridize to a target nucleic acid andinduce a desired effect. “Targeted to” means having a nucleobasesequence that will allow hybridization to a target nucleic acid toinduce a desired effect. In certain embodiments, a desired effect isreduction of a target nucleic acid.

As used herein, the term “variant” refers to a polynucleotide (orpolypeptide) having a sequence substantially similar to a referencepolynucleotide (or polypeptide). In the case of a polynucleotide, avariant can have deletions, substitutions, additions of one or morenucleotides at the 5′ end, 3′ end, and/or one or more internal sites incomparison to the reference polynucleotide. Similarities and/ordifferences in sequences between a variant and the referencepolynucleotide can be detected using conventional techniques known inthe art, for example polymerase chain reaction (PCR) and hybridizationtechniques. Variant polynucleotides also include synthetically derivedpolynucleotides, such as those generated, for example, by usingsite-directed mutagenesis. Generally, a variant of a polynucleotide,including, but not limited to, a DNA, can have at least about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99% or more sequence identity to thereference polynucleotide as determined by sequence alignment programsknown by skilled artisans. In the case of a polypeptide, a variant canhave deletions, substitutions, additions of one or more amino acids incomparison to the reference polypeptide. Similarities and/or differencesin sequences between a variant and the reference polypeptide can bedetected using conventional techniques known in the art, for exampleWestern blot. Generally, a variant of a polypeptide, can have at leastabout 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99% or more sequence identity to thereference polypeptide as determined by sequence alignment programs knownby one of ordinary skill in the art.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any elements, steps, or ingredients notspecified in the claimed composition or method. As used herein,“consisting essentially of” does not exclude materials or steps that donot materially affect the basic and novel characteristics of the claimedcomposition or method. Any recitation herein of the term “comprising”,particularly in a description of components of a composition or in adescription of steps of a method, is understood to encompass thosecompositions and methods consisting essentially of and consisting of therecited components or steps.

In some embodiments of the methods or processes described herein, thesteps can be carried out in any order, except when a temporal oroperational sequence is explicitly recited. Furthermore, in someembodiments, the specified steps can be carried out concurrently unlessexplicit claim language recites that they be carried out separately. Forexample, in some embodiments a claimed step of doing X and a claimedstep of doing Y can be conducted simultaneously within a singleoperation, and the resulting process will fall within the literal scopeof the claimed process.

The section headings, e.g., (a), (b), (i) etc., are presented merely forease of reading the specification and claims, as they are used hereinfor organizational purposes only and are not to be construed as limitingthe subject matter described. The use of headings in the specificationor claims does not require the steps or elements be performed inalphabetical or numerical order or the order in which they arepresented. All documents, or portions of documents, cited in theapplication including, without limitation, patents, patent applications,articles, books, manuals, and treatises are hereby expresslyincorporated by reference in their entireties.

As will be understood by one having ordinary skill in the art, for anyand all purposes, such as in terms of providing a written description,all ranges disclosed herein also encompass any and all possiblesub-ranges and combinations of sub-ranges thereof. Any listed range canbe easily recognized as sufficiently describing and enabling the samerange being broken down into at least equal halves, thirds, quarters,fifths, tenths, etc. As a non-limiting example, each range discussedherein can be readily broken down into a lower third, middle third andupper third, etc. As will also be understood by one skilled in the artall language such as “up to,” “at least,” “greater than,” “less than,”and the like include the number recited and refer to ranges which can besubsequently broken down into sub-ranges as discussed above. Finally, aswill be understood by one skilled in the art, a range includes eachindividual member. Thus, for example, a group having 1-3 articles refersto groups having 1, 2, or 3 articles. Similarly, a group having 1-5articles refers to groups having 1, 2, 3, 4, or 5 articles, and soforth.

I. Cardiac Diseases and Micro-Ribonucleic Acid (MIRNA)

Cardiac disease or heart disease is a disease for which several classesor types exist (e.g., Ischemic Cardiomyopathy (ICM), DilatedCardiomyopathy (DCM), Aortic Stenosis (AS)) and, many require uniquetreatment strategies. Thus, heart disease is not a single disease, butrather a family of disorders arising from distinct cell types (e.g.,myocardial cells) by distinct pathogenetic mechanisms. The challenge ofheart disease treatment has been to target specific therapies toparticular heart disease types, to maximize effectiveness and tominimize toxicity. Improvements in heart disease categorization(classification) have thus been central to advances in heart diseasetreatment. As used herein, cardiac disease encompasses the followingnon-limiting examples: heart failure (e.g., congestive heart failure),ischemic cardiomyopathy, hypertrophic cardiomyopathy, restrictivecardiomyopathy, alcoholic cardiomyopathy, viral cardiomyopathy,tachycardia-mediated cardiomyopathy, stress-induced cardiomyopathy,amyloid cardiomyopathy, arrhythmogenic right ventricular dysplasia, leftventricular noncompaction, endocardial fibroelastosis, aortic stenosis,aortic regurgitation, mitral stenosis, mitral regurgitation, mitralprolapse, pulmonary stenosis, pulmonary regurgitation, tricuspidstenosis, tricuspid regurgitation, congenital disorder, geneticdisorder, or a combination thereof.

Heart cell regeneration: Throughout the 20th century the human heart wasbelieved to be a terminally differentiated post mitotic organ, unable tobe repaired after an injury. This was challenged in 2001 when mitosis incardiomyocytes was evident after a myocardial infarction. Studies byothers confirmed that adult mammalian hearts can elicit a primitiveregeneration response upon injury with mature differentiated mononuclearmammalian cardiomyocytes re-entering the cell cycle upon application ofchemical compounds that target specific signaling pathways.

miRNAs (also referred to as miRs) are small non-coding RNA moleculesconserved in plants, animals, and some viruses, which function in RNAsilencing and post-transcriptional regulation of gene expression.Identified in 1993, they are a vital and evolutionarily component ofgenetic regulation. They function via base-pairing and silencingcomplementary sequences within mRNA molecules thereby modulating targetprotein expression and downstream signaling pathways. There are 1000known miRs in the human genome that can target 60% of human genes. Inanimals, miRNAs are processed from larger primary transcripts (pri-miRNAor pri-miR) through an approximate 60-bp hairpin precursor (pre-miRNA orpre-miR) into the mature forms (miRNA) by two RNAse III enzymes Droshaand Dicer. The mature miRNA is loaded into the 50 ribonucleoproteincomplex (RISC), where it typically guides the downregulation of targetmRNA through base pair inter-actions. Pri-miRNAs are transcribed by RNApolymerase II and predicted to be regulated by transcription factors inan inducible manner. While some miRNAs show ubiquitous expression,others exhibit only limited developmental stage-, tissue- or celltype-specific patterns of expression.

As described in greater detail below, measurements previously made inmyocardial tissue have suggested the miRNAs play a regulatory role inmyocardial growth, fibrosis, and remodeling. In particular, ribonucleicacid interference (RNAi) technology is an area of intense research forthe development of new therapies for heart disease, with studiesdemonstrating the utility of adeno-associated virus (AAV) for deliveringoligonucleotides in vivo. Two separate AAV2/9 virus' expressingantagonists of microRNAs (miRs) let-7a/let-c and miR-99/100 can induceproliferation of cardiomyocytes in the ischemic mouse heart for up to 3months following a single injection. Transcriptomic and translationalanalysis on mice heart cells and tissues treated with viral deliveredmiR antagonists showed differences in the expression of genes andproteins involved in cardiac development, proliferation and musclestructure and function, implying that a similar regenerative effect,through targeting of these miRs, may occur in human cardiac myocytes andmodels of DMD.

RNAi technology can take many forms, but it is typically implementedwithin a cell in the form of a base-pair short hairpin (sh) RNA (shRNA),which is processed into an approximately 20 base pair small interferingRNA through the endogenous miR pathway. Viral delivery of complementarysequences to miRs is a common approach. AAV vectors are optimal incardiovascular muscle gene delivery since they a) contain no viralprotein-coding sequences to stimulate an immune response, b) do notrequire active cell division for expression to occur and c) have asignificant advantage over adenovirus vectors because of their stable,long-term expression of recombinant genes in myocytes in vivo. Viraldelivery of genes are in development for the treatment of DMD andinclude AAV1-gamma-sarcoglycan vector as a therapy for LGMD, recombinant(r) AAV2.5 vector for delivery of mini dystrophin, and rAAV, rhesusserotype 74.

As described herein, the mechanism by which the miRNA antagonistfunctions to inhibit the activity of the target miRNA is not limited inany way. For example, a nucleic acid-based antagonist, in someembodiments, may form a duplex with the target miRNA sequences andprevent proper processing of the mature miRNA product from itsprecursor, or may prevent the mature miRNA from binding to its targetgene, or may lead to degradation of pri-, pre-, or mature miRNA, or mayact through some other mechanism.

let-7a/c and miR-100/99: By studying the mechanisms of heartregeneration in zebrafish and neonatal mice, scientists have found thatheart regeneration is a primarily cardiomyocyte-mediated process thatoccurs by dedifferentiation of mature cardiomyocytes followed byproliferation and further re-differentiation. Epigenetic remodeling andcell cycle control are two key steps controlling this regenerativeprocess. Aguirre et al (Cell Stem Cell. 2014; 15(5):589-604) reported avery relevant study, which investigated the underlying mechanism ofheart regeneration and identified a series of miRs strongly involved inzebrafish heart regeneration. Focus on those miRs that presentsignificant expression changes and that were conserved acrossvertebrates, both in sequence and 3′ UTR binding sites, led to theidentification of two miR families (miR-99/100, let-7a/c) clustered intwo well-defined genomic locations. This finding was supported by acommon role for the miR-99a/Let-7c-5p cluster in regulating vertebratecardiomyogenesis. MIRANDA-based miR-UTR binding predictions showed astrong interaction for miR-99/100 with zebrafish FNTβ (beta subunit offarnesyl-transferase) and SMARCA5 (SWI/SNF-related matrix associatedactin-dependent regulator of chromatin subfamily a, member 5), linkingthe miR families to cell cycle and epigenetic control in cardiomyocytes.Interestingly, miR-99/100 and let-7a/c levels are low during earlymammalian heart development and promote quick cardiac mass growth, butincrease exponentially during late development, with a correspondingdecrease in FNTβ and SMARCA5 protein levels to block furthercardiomyocyte proliferation. Postmortem analysis of injured human hearttissue, suggests that these miRs constitute a conserved roadblock tocardiac regeneration in adults. RNA-seq transcriptomic analysis onneonatal mouse cardiomyocytes transduced two viral delivered antagoniststo let-7a/c and miR-99/100 revealed differences in genes involved inepigenetic remodeling, demethylation, cardiac development,proliferation, and unexpectedly, metabolic pathways and musclestructural and function. Indeed, miR-let 7a/c and miR-99/100 inhibitiontargets 1072 and 47 genes, respectively.

A number of selective genes involved in muscle structure and functioninclude actin/myosin, NFKB inhibitor interacting Ras-like 2,sarcoglycan, delta (35 kDa dystrophin-associated glycoprotein) and IGF-1that are current therapeutic targets for muscular dystrophies (see,Table 1 below). Semi-quantitative mass spectrometry on organ cultures ofmouse hearts treated with the inhibitors identified metabolic andmitochondrial processes as key actors, while also highlighting changesin cytoskeleton and proteins involved in muscle contraction as shown inTable 2.

TABLE 1 Non-limiting examples of selected target genes for Let-7a-5p inneonatal mouse cardiac myocytes Target Representative gene transcriptGene name IGF2BP3 NM_006547 insulin-like growth factor 2 mRNA bindingprotein 3 COL24A1 NM_152890 collagen, type XXIV, alpha 1 TMEM135NM_001168724 transmembrane protein 135 CHD9 NM_025134 chromodomainhelicase DNA binding protein 9 IGF1R NM_000875 insulin-like growthfactor 1 receptor IGF2BP2 NM_001007225 insulin-like growth factor 2 mRNAbinding protein 2 ACTA1 NM_001100 actin, alpha 1, skeletal muscle FKBP10NM_021939 FK506 binding protein 10, 65 kDa ACVR1B NM_004302 activin Areceptor, type IB MYO5B NM_001080467 myosin VB INSR NM_000208 insulinreceptor ITGB8 NM_002214 integrin, beta 8 FRS2 NM_001042555 fibroblastgrowth factor receptor substrate 2 ACVR1C NM_001111031 activin Areceptor, type IC COL3A1 NM_000090 collagen, type III, alpha 1 NGFNM_002506 nerve growth factor (beta polypeptide) COL4A6 NM_001847collagen, type IV, alpha 6 CTHRC1 NM_138455 collagen triple helix repeatcontaining 1 IRS2 NM_003749 insulin receptor substrate 2 NOS1 NM_000620nitric oxide synthase 1 (neuronal) MYRIP NM_015460 myosin VIIA and Rabinteracting protein COL11A1 NM_001190709 collagen, type XI, alpha 1NKIRAS2 NM_001001349 NFKB inhibitor interacting Ras-like 2 SMAD2NM_001003652 SMAD family member 2 TTL NM_153712 tubulin tyrosine ligaseSGCD NM_000337 sarcoglycan, delta (35 kDa dystrophin- associatedglycoprotein) COL14A1 NM_021110 collagen, type XIV, alpha 1 COL1A1NM_000088 collagen, type I, alpha 1 COL15A1 NM_001855 collagen, type XV,alpha 1 FNDC3B NM_001135095 fibronectin type III domain containing 3BCOL4A5 NM_000495 collagen, type IV, alpha 5 MFAP3L NM_001009554microfibrillar-associated protein 3-like ACVR2B NM_001106 activin Areceptor, type IIB RPS6KA3 NM_004586 ribosomal protein S6 kinase, 90kDa, polypeptide 3

II. Muscular Dystrophy

The muscular dystrophies (MD) are a group of more than 30 geneticdiseases characterized by progressive weakness and degeneration of theskeletal muscles that control movement. Some forms of MD are seen ininfancy or childhood, while others may not appear until middle age orlater. The disorders differ in terms of the distribution and extent ofmuscle weakness (some forms of MD also affect cardiac muscle), age ofonset, rate of progression, and pattern of inheritance.

Duchenne muscular dystrophy (DMD) is a progressive, an X-linkedrecessive inherited muscle-wasting disease, leading to severe disabilityand premature death. DMD is caused by mutations on one of the 21.2 bandon the short arm of the X chromosomes, affecting half of the maleinfants of mothers who carry the genetic defect. This gene isresponsible for producing cytoplasmic dystrophin protein, an essentialpart of a protein complex that connects the cytoskeleton of a musclefiber to the surrounding extracellular matrix through the cell membrane.Without dystrophin, muscles degenerate. The primary symptoms of thedisease is muscle weakness, respiratory problems and early diastolicdysfunction caused by focal fibrosis which proceeds to dilatedcardiomyopathy (DCM), complicated by heart failure and arrhythmia inmost patients. Current treatments for DMD are solely symptomatic. Table3 below provides a listing of non-limiting examples of current standardapproaches for the treatment of Duchenne Muscular Dystrophy.

TABLE 3 Standard therapy for muscular Duchenne dystrophy (DMD)Intervention Timing/Use Examples/ Limitations Corticosteroids Age < 4Prednisone, Prednisolone, Deflazacort/ years Behavioral changes, failureto gain height, weight gain, osteoporosis, impaired glucose tolerance,blood pressure changes, immune/adrenal suppression, dyspepsia/pepticulceration, cataract, and skin changes, cushingoid features, red reflexof eyes, bone fractures, infections. Nutrition Age < 4 Calcium andvitamin D intake, years controlled sodium intake/ Weight control.Respiratory With Ventilators/ Management of chest care symptomsinfections with antibiotics. Cardiac care Age 5-10 ACE-inhibitors,beta-blockers, diuretics years with onset of HF. Anticoagulation therapyconsidered with severe cardiac dysfunction to prevent systemicthromboembolic events. If ventricular arrhythmias occur, antiarrhythmictreatment is introduced with possible negative inotropic effects.Echocardiogram and ECG every five years. Orthopedics Variable Splinting,Knee-Ankle-Foot Orthosis, fusion. Psychosocial Variable Social(information, advocacy and advice) and psychological supportRehabilitation At Physiotherapists and occupational diagnosistherapists. Moderate active exercise.

There have been over 200 clinical studies for DMD and selectiveinterventional studies with results. Table 4 below provides a listing ofnon-limiting examples of Selective Clinical Investigative therapies forinterventional DMD trials that have been reported previously. CurrentDMD therapeutic approaches in clinical development include, 1) genedelivery therapy aimed at reintroducing a functional recombinant versionof the dystrophin gene, 2) exon skipping, 3) read-through strategies fornonsense mutations, 4) cell-based therapies, 5) utrophin upregulation,myostatin inhibition, Insulin like growth factor-1 (IGF-1) expression,6) approved commercial products and 7)anti-Inflammatories/anti-oxidants.

TABLE 4 Selective clinical investigative agents interventional DMDtrials for which results have been previously reported Clinical TrialsTherapy Phase Identifier: Main findings/status Idebenone IIa NCT00654784Respiratory treatment effect III NCT01027884 on peak expiratory flow (p= 0.039 for PEF) [62]. Reduced the loss of respiratory function.Eplerenone II NCT01521546 Lower decline in left ventricularcircumferential strain than placebo VECTTOR NCT01874275 Indicted in theU.S. for chronic, intractable pain and post-surgical trauma pain. Deviceconsidered for off- label use. AVI-4658 II NCT01396239 Increase in sixminute walk AVI-4658 I/II NCT00844597 test, decreased incidence in lossof ambulation [66-67]. New dystrophin protein expression (p = 0 · 0203).Restoration of α-sarcoglycan and nitric oxide synthase. Ataluren/ IINCT00264888 Approval in European Union PTC124/ III NCT00592553 approvalfor nonsense Translarna mutation DMD. DMN044/ I/II NCT01037309 Increasein expression of PRO044 dystrophin protein. CAT-1004 I/II NCT02439216Phase I showed safety, and no adverse events rAAV2.5- I NCT00428935Failure to establish long- CMV- term transgene expression minidystrophinin muscle fibers. Glutamine III NCT00296621 No disease modifying effect.NCT00018109 No disease modifying effect.

Major problems of finding effective treatments is the need to targetdifferent muscles in the body, the requirement of a long-term effect,the problem of fibrosis and the necessity for various versions of a drugto address different mutations. Although long-term expression could beachieved with gene therapy, restoration of dystrophin protein expressionis complicated by the large size of dystrophin cDNA that cannot becarried by a viral vector. Smaller versions of dystrophin (mini- andmicro-dystrophins) have been developed to address this problem. Forlimb-girdle muscular dystrophy type 2D (LGMD) clinical trials have shownpromising results. Genome editing through the CRISPR/Cas9 system hasdemonstrated encouraging findings in preclinical murine models but isnot yet possible in humans.

Despite a number of investigative therapies, full recovery of dystrophinprotein is not achievable. Finding alternative therapeutic strategiesthat increase the expression of compensatory genes and proteins andregenerate both endogenous cardiac muscle in DMD patients is an urgentnecessity.

Intriguingly, heart regenerating vertebrates that do not developpathologic remodeling after a heart attack (including neonatal mice),heal by cardiomyocyte dedifferentiation and proliferation, illustratingtwo important facts: 1) cardiomyocytes represent a larger and moreefficient pool of regenerative precursors than stem cells and 2)regeneration is an innate property of mammalian hearts and can lead tofunctional recovery, albeit inefficiently, in adults.

III. Compositions of the Disclosure MicroRNA Antagonists

Disclosed herein includes embodiments of compositions that include aplurality of microRNA (miR) antagonists. As used herein, “miRantagonist” refers to an agent designed to interfere with or inhibit theactivity of a miRNA. In certain embodiments, a miR antagonist comprisesan antisense compound targeted to a miRNA. In certain embodiments, a miRantagonist comprises a modified oligonucleotide having a nucleotidesequence that is complementary to the nucleotide sequence of a miRNA, ora precursor thereof. In other embodiments, a miR antagonist comprises asmall molecule, or the like that interferes with or inhibits theactivity of a miRNA. In some embodiments, a miR antagonist is a miR-99aantagonist. In some embodiments, a miR antagonist is a miR-100-5pantagonist. In some embodiments, a miR antagonist is a miR-Let-7a-5pantagonist. In some embodiments, a miR antagonist is a miR-Let-7c-5pantagonist. The miR antagonists disclosed herein are useful, forexample, in providing compositions and methods to prevent, inhibit, orreduce target gene expression in, for example, myocardium (e.g.,myocardial tissue, myocardial cells). Thus, some of the embodimentsdisclosed herein relate to the use of the miR antagonists of thedisclosure in methods for evaluation and therapy of cardiac diseases,including heart failure.

Implementations of embodiments of the compositions according to thisaspect and other aspects of the disclosure can include one or more ofthe following features. In some embodiments, the plurality of miRantagonists includes 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 miRantagonists or a number of antagonists that is within a range defined byany two of the aforementioned values. In some embodiments, the pluralityof miR antagonists includes one or more selected from miR-99aantagonists, miR-100-5p antagonists, miR-Let-7a-5p antagonists,miR-Let-7c-5p antagonists, and combinations thereof. In someembodiments, the plurality of miR antagonists includes one or moremiR-99a antagonists, one or more miR-100-5p antagonists, one or moremiR-Let-7a-5p antagonists, and one or more miR-Let-7c-5p antagonists. Insome embodiments, the numbers of each miR antagonist group are the samein the plurality of miR antagonists. In some embodiments, the numbers ofeach miR antagonist group are not the same in the plurality of miRantagonists.

Accordingly, in some embodiments, the plurality of miR antagonistsincludes at least one miR antagonist comprising a nucleotide sequencehaving, or having about, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 100%, or a range between any two of these values, sequenceidentity to one or more of the miR antagonists disclosed herein. Forexample, in some embodiments, the miR antagonist comprises, or consistsof, a nucleotide sequence having at least about 85%, at least about 90%,at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99%, or more, sequence identity to one or moreof the miR antagonists disclosed herein. In some embodiments, the miRantagonist comprises, or consists of, a nucleotide sequence having atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, ormore, sequence identity to one or more of the miR antagonists disclosedherein. In some embodiments, the miR antagonist comprises, or consistsof, a nucleotide sequence having about 85%, about 90%, about 95%, about96%, about 97%, about 98%, about 99%, about 100%, or a range between anytwo of these values, sequence identity to one or more of the miRantagonists disclosed herein.

In some embodiments, at least one of the one or more miR-99a antagonistsincludes an anti-miR-99a comprising a nucleotide sequence having atleast about, or having about, 80%, 85%, 90%, 95%, 96%, 97, 98%, 99% or100%, or a range between any two of these values, sequence identity to asequence selected from the group consisting of SEQ ID NOs 47, 48, 50,52, and 54. In some embodiments, at least one of the one or moremiR-100-5p antagonists includes an anti-miR-100-5p comprising anucleotide sequence having at least about, or having about, 80%, 85%,90%, 95%, 96%, 97, 98%, 99% or 100%, or a range between any two of thesevalues, sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs 46, 49, 51, 53, and 55. In some embodiments, atleast one of the one or more Let-7a-5p antagonists includes ananti-miR-Let-7a-5p comprising a nucleotide sequence having at leastabout, or having about, 80%, 85%, 90%, 95%, 96%, 97, 98%, 99% or 100%,or a range between any two of these values, sequence identity to asequence selected from the group consisting SEQ ID NOs: 37, 39, and40-45. In some embodiments, at least one of the one or more Let-7c-5pantagonists includes an anti-miR-Let-7c-5p comprising a nucleotidesequence having at least about, or having about, 80%, 85%, 90%, 95%,96%, 97, 98%, 99% or 100%, or a range between any two of these values,sequence identity to a sequence selected from the group consisting ofSEQ ID NOs: 36, 38, and 40-45.

In some embodiments of the compositions disclosed herein, one or more ofthe followings applies. In some embodiments, at least one of the one ormore miR-99a antagonists includes an anti-miR-99a comprising anucleotide sequence having one or more mismatched nucleobases withrespect to a sequence selected from the group consisting of SEQ ID NOs:47, 48, 50, 52, and 54. In some embodiments, at least one of the one ormore miR-100-5p antagonists includes an anti-miR-100-5p comprising anucleotide sequence having one or more mismatched nucleobases withrespect to a sequence selected from the group consisting of SEQ ID NOs:46, 49, 51, 53, and 55. In some embodiments, at least one of the one ormore Let-7a-5p antagonists includes an anti-miR-Let-7a-5p comprising anucleotide sequence having one or more mismatched nucleobases withrespect to a sequence selected from the group consisting of SEQ ID NOs:37, 39, and 40-45. In some embodiments, at least one of the one or moreLet-7c-5p antagonists includes an anti-miR-Let-7c-5p comprising anucleotide sequence having one or more mismatched nucleobases withrespect to a sequence selected from the group consisting of SEQ ID NOs:36, 38, and 40-45.

In some embodiments, the plurality of miR antagonists includes at leastone miR antagonist comprising a nucleotide sequence having, or havingabout, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or a range between any two ofthese values, mismatched nucleobases with respect to the nucleotidesequence of one or more of the miR antagonists disclosed herein. Forexample, in some embodiments, the miR antagonist comprises, or consistsof, a nucleotide sequence having at least about 1, at least about 2, atleast about 3, at least about 4, at least about 5, or more, mismatchednucleobases with respect to the nucleotide sequence of one or more ofthe miR antagonists disclosed herein. In some embodiments, the miRantagonist comprises, or consists of, a nucleotide sequence having atleast about 6, at least about 7, at least about 8, at least about 9, atleast about 10, or more, mismatched nucleobases with respect to thenucleotide sequence of one or more of the miR antagonists disclosedherein.

Accordingly, in some embodiments, at least one of the one or moremiR-99a antagonists includes an anti-miR-99a comprising a nucleotidesequence having, or having about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or arange between any two of these values, mismatched nucleobases withrespect to a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 47, 48, 50, 52, and 54. In some embodiments, at least one ofthe one or more miR-100-5p antagonists includes an anti-miR-100-5pcomprising a nucleotide sequence having, or having about, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or a range between any two of these values, mismatchednucleobases with respect to a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 46, 49, 51, 53, and 55. In someembodiments, at least one of the one or more Let-7a-5p antagonistsincludes an anti-miR-Let-7a-5p comprising a nucleotide sequence having,or having about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or a range between anytwo of these values, mismatched nucleobases with respect to a nucleotidesequence selected from the group consisting of SEQ ID NOs: 37, 39, and40-45. In some embodiments, at least one of the one or more Let-7c-5pantagonists includes an anti-miR-Let-7c-5p comprising a nucleotidesequence having, or having about, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or arange between any two of these values, mismatched nucleobases withrespect to a nucleotide sequence selected from the group consisting ofSEQ ID NOs: 36, 38, and 40-45.

In various embodiments of the compositions disclosed herein, at leastone of the anti-miRs includes one or more chemical modificationsdescribed herein. Suitable chemical modifications include, but are notlimited to, modifications to a nucleobase, a sugar, and/or aninternucleoside linkage. A modified nucleobase, sugar, and/orinternucleoside linkage may be selected over an unmodified form becauseof desirable properties such as, for example, enhanced cellular uptake,enhanced affinity for other oligonucleotides or nucleic acid targets andincreased stability in the presence of nucleases. Accordingly, in someembodiments of the compositions disclosed herein, at least one of theanti-miRs includes one or more chemical modifications selected from thegroup consisting of a modified internucleoside linkage, a modifiednucleotide, and a modified sugar moiety, and combinations thereof.

In some embodiments, the one or more chemical modifications includes amodified internucleoside linkage. Generally, a modified internucleosidelinkage can be any internucleoside linkage known in the art.Non-limiting examples of suitable modified internucleoside linkageinclude a phosphorothioate, 2′-Omethoxyethyl (MOE), 2′-fluoro,alkylphosphonate, phosphorodithioate, alkylphosphonothioate,phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate,carboxymethyl ester, and combinations thereof. In some embodiments, themodified internucleoside linkage comprises a phosphorus atom. In someembodiments, the modified internucleoside linkage does not comprise aphosphorus atom. In certain such embodiments, an internucleoside linkageis formed by a short chain alkyl internucleoside linkage. In certainsuch embodiments, an internucleoside linkage is formed by a cycloalkylinternucleoside linkages. In certain such embodiments, aninternucleoside linkage is formed by a mixed heteroatom and alkylinternucleoside linkage. In certain such embodiments, an internucleosidelinkage is formed by a mixed heteroatom and cycloalkyl internucleosidelinkages. In certain such embodiments, an internucleoside linkage isformed by one or more short chain heteroatomic internucleoside linkages.In certain such embodiments, an internucleoside linkage is formed by oneor more heterocyclic internucleoside linkages. In certain suchembodiments, an internucleoside linkage has an amide backbone. Incertain such embodiments, an internucleoside linkage has mixed N, O, Sand CH₂ component parts. In some embodiments, at least one of theanti-miRs includes a modified internucleoside linkage which is aphosphorothioate internucleoside linkage.

In some embodiments, at least one of the one or more chemicalmodifications includes a modified nucleotide. A modified nucleotide cangenerally be any modified nucleotide and can be for example, a lockednucleic acid (LNA) chemistry modification, a peptide nucleic acid (PNA),an arabino-nucleic acid (FANA), an analogue, a derivative, or acombination thereof. In some embodiments, the modified nucleotidecomprises 5-methylcytosines. In some embodiments, a modified nucleotideis selected from 5-hydroxymethyl cytosine, 7-deazaguanine and7-deazaadenine. In certain embodiments, the modified nucleotide isselected from 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and2-pyridone. In certain embodiments, the modified nucleotide is selectedfrom 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2 aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. In certain embodiments, a modified nucleotidecomprises a polycyclic heterocycle. In certain embodiments, a modifiednucleotide comprises a tricyclic heterocycle. In certain embodiments, amodified nucleotide comprises a phenoxazine derivative. In certainembodiments, the phenoxazine can be further modified to form anucleobase known in the art as a G-clamp.

In some embodiments, the modified nucleotide includes a locked nucleicacid (LNA). In some embodiments, the one or more chemical modificationsincludes at least one locked nucleic acid (LNA) chemistry modificationsto enhance the potency, specificity and duration of action and broadenthe routes of administration of oligonucleotides. This can be achievedby substituting some of the nucleobases in a base nucleotide sequence byLNA nucleobases. The LNA modified nucleotide sequences may have a sizesimilar to the parent nucleobase or may be larger or preferably smaller.In some embodiments, the LNA-modified nucleotide sequences contain lessthan about 70%, less than about 65%, more preferably less than about60%, less than about 55%, most preferably less than about 50%, less thanabout 45% LNA nucleobases and that their sizes are between about 5 and25 nucleotides, more preferably between about 12 and 20 nucleotides. Insome embodiments, the locked nucleic acid (LNA) is incorporated at oneor both ends of the modified anti-miR.

In some embodiments, the one or more chemical modifications include atleast one modified sugar moiety. In some embodiments, In certainembodiments, a sugar modified nucleoside is a 2′-modified nucleoside,wherein the sugar ring is modified at the 2′ carbon from natural riboseor 2′-deoxy-ribose. In some embodiments, a 2′-modified nucleoside has abicyclic sugar moiety. In certain such embodiments, the bicyclic sugarmoiety is a D sugar in the alpha configuration. In certain suchembodiments, the bicyclic sugar moiety is a D sugar in the betaconfiguration. In certain such embodiments, the bicyclic sugar moiety isan L sugar in the alpha configuration. In certain such embodiments, thebicyclic sugar moiety is an L sugar in the beta configuration.

In some embodiments, the bicyclic sugar moiety comprises a bridge groupbetween the 2′ and the 4′-carbon atoms. In certain such embodiments, thebridge group comprises from 1 to 8 linked biradical groups. In certainembodiments, the bicyclic sugar moiety comprises from 1 to 4 linkedbiradical groups. In certain embodiments, the bicyclic sugar moietycomprises 2 or 3 linked biradical groups. In certain embodiments, thebicyclic sugar moiety comprises 2 linked biradical groups. In certainembodiments, a linked biradical group is selected from —O—, —S—,—N(R₁)—, —C(R₁)(R₂)—, —C(R₁)═C(R₁)—, —C(R₁)═N—, —C(═NR₁)—, —Si(R₁)(R₂)—,—S(═O)₂—, —S(═O)—, —C(═O)— and —C(═S)—; where each R₁ and R₂ is,independently, H, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl,C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substitutedC₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, a heterocycleradical, a substituted heterocycle radical, heteroaryl, substitutedheteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclicradical, halogen, substituted oxy (—O—), amino, substituted amino,azido, carboxyl, substituted carboxyl, acyl, substituted acyl, CN,thiol, substituted thiol, sulfonyl (S(═O)₂—H), substituted sulfonyl,sulfoxyl (S(═O)—H) or substituted sulfoxyl; and each substituent groupis, independently, halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl,C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substitutedC₂-C₁₂ alkynyl, amino, substituted amino, acyl, substituted acyl, C₁-C₁₂aminoalkyl, C₁-C₁₂ aminoalkoxy, substituted C₁-C₁₂ aminoalkyl,substituted C₁-C₁₂ aminoalkoxy or a protecting group.

In some embodiments, the bicyclic sugar moiety is bridged between the 2′and 4′ carbon atoms with a biradical group selected from —O—(CH₂)_(p)—,—O—CH₂—, —O—CH₂CH₂—, —O—CH(alkyl)-, —NH—(CH₂)_(p)—,—N(alkyl)-(CH₂)_(p)—, —O—CH(alkyl)-, —(CH)alkyl))—(CH₂)_(p)—,—NH—O—(CH₂)_(p)—, —N(alkyl)-O—(CH₂)_(p)—, or —O—N(alkyl)-(CH₂)_(p)—,wherein p is 1, 2, 3, 4 or 5 and each alkyl group can be furthersubstituted. In certain embodiments, p is 1, 2 or 3.

In some embodiments, a 2′-modified nucleoside comprises a 2′-substituentgroup selected from halo, allyl, amino, azido, SH, CN, OCN, CF₃, OCF₃,O—, S—, or N(R_(m))-alkyl; O—, S—, or N(R_(m))-alkenyl; O—, S— orN(R_(m))-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl,O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)) orO—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl. These 2′-substituent groups can be furthersubstituted with one or more substituent groups independently selectedfrom hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂),thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

In some embodiments, a 2′-modified nucleoside comprises a 2′-substituentgroup selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂, CH₂—CH═CH₂,O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)),—O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substituted acetamide(O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R_(n) is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In some embodiments, a 2′-modified nucleoside comprises a 2′-substituentgroup selected from F, OCF3, O—CH3, OCH2CH2OCH3, 2′-O(CH2)2SCH3,O—(CH2)2-O—N(CH3)2, O(CH2)2O(CH2)2N—(CH3)2, and O—CH2-C(═O)—N(H)CH3.

In some embodiments, a 2′-modified nucleoside comprises a 2′-substituentgroup selected from F, O—CH₃, and OCH₂CH₂OCH₃.

In some embodiments, a sugar-modified nucleoside is a 4′-thio modifiednucleoside. In certain embodiments, a sugar-modified nucleoside is a4′-thio-2′-modified nucleoside. A 4′-thio modified nucleoside has aβ-D-ribonucleoside where the 4′-O replaced with 4′-S. A4′-thio-2′-modified nucleoside is a 4′-thio modified nucleoside havingthe 2′-OH replaced with a 2′-substituent group. Suitable 2′-substituentgroups include 2′-OCH₃, 2′-O—(CH₂)₂—OCH₃, and 2′-F.

Accordingly, in some embodiments of the disclosure, the modified sugarmoiety is a 2′-O-methoxyethyl modified sugar moiety, a 2′-methoxymodified sugar moiety, a 2′-O-alkyl modified sugar moiety, a bicyclicsugar moiety, or a combination thereof. In some embodiments, themodified sugar moiety comprises a 2′-O-methyl sugar moiety.

Expression Cassettes

In some embodiments, one or more of the miR antagonists described hereinare encoded by and expressed from expression cassettes. Thus, in oneaspect, some embodiments of the present disclosure related to expressioncassettes that include a nucleotide sequence encoding one or more miRantagonists described herein. As used herein, “expression” refers to theprocess of converting genetic information of a polynucleotide into RNAthrough transcription, which is typically catalyzed by an enzyme, RNApolymerase, and, where the RNA encodes a polypeptide, into protein,through translation of mRNA on ribosomes to produce the encoded protein.The term “expression cassette” as used herein, refers to a nucleic acidconstruct that encodes a gene, a protein, or a functional RNA operablylinked to expression control elements, such as a promoter, andoptionally, any or a combination of other nucleic acid sequences thataffect the transcription or translation of the gene, such as, but notlimited to, a transcriptional terminator, a ribosome binding site, asplice site or splicing recognition sequence, an intron, an enhancer, apolyadenylation signal, an internal ribosome entry site, etc.

Cloning Vectors and Expression Vectors

In a related aspect, one or more of the miR antagonists described hereincan be encoded by and/or expressed from a cloning vector or anexpression vector. Accordingly, some embodiments of the presentapplication are directed to a cloning vector or expression vector thatincludes an expression cassette as disclosed herein. As used herein, theterm “vector” refers to a nucleic acid construct, typically a plasmid ora virus, used to transmit genetic material to a host cell. Vectors canbe, for example, viruses, plasmids, cosmids, or phage. A vector as usedherein can be composed of either DNA or RNA. In some embodiments, avector is composed of DNA. In some embodiments, a vector is composed ofRNA. The term “vector” includes cloning vectors and expression vectors,as well as viral vectors and integrating vectors. An “expression vector”is a vector that is capable of directing the expression of a gene, orprotein encoded by one or more genes carried by the vector when it ispresent in the appropriate environment. Vectors are preferably capableof autonomous replication. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and a geneis said to be “operably linked to” the promoter.

Accordingly, in some embodiments, the cloning vector or expressionvector disclosed herein includes an expression cassette including anucleotide sequence which encodes one or more miR antagonists describedherein. In some embodiments, the cloning vector or expression vectordisclosed herein includes an expression cassette including a nucleotidesequence which encodes one or more miR-99a antagonists, one or moremiR-100-5p antagonists, one or more miR-Let-7a-5p antagonists, and oneor more miR-Let-7c-5p antagonists.

In some embodiments, the cloning vector or expression vector is a viralvector. As used herein, a “viral vector” is a viral-derived nucleic acidmolecule that is capable of transporting another nucleic acid into acell. A viral vector is capable of directing expression of a gene, aprotein or proteins encoded by one or more genes carried by the vectorwhen it is present in the appropriate environment. Examples for viralvectors include, but are not limited to retroviral vectors, adenoviralvectors, lentiviral vectors, and adeno-associated viral vectors.

Accordingly, in some embodiments, the viral vector is a lentiviralvector or an adeno-associated viral (AAV) vector or any serotype. Asused herein, the term “serotype” or “serovar” is a distinct variationwithin a species of bacteria or virus or among immune cells of differentindividuals. These microorganisms, viruses, or cells are classifiedtogether based on their cell surface antigens, allowing theepidemiologic classification of organisms to the sub-species level.Generally, the AAV vector can be any existing AAV vectors and can be,for example, an AAV vector selected from the group consisting ofserotype 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 or chimeric AAV derivedthereof, which will be even better suitable for high efficiencytransduction in the tissue of interest. Upon transfection, AAV elicitsonly a minor immune reaction (if any) in the host. Therefore, AAV vectoris highly suited for gene therapy approaches. It has been reported that,for transduction in mice, AAV serotype 6 and AAV serotype 9 areparticularly suitable. For gene transfer into a human, AAV serotypes 1,6, 8 and 9 are generally preferred. It has been also assumed that thecapacity of AAV for packaging a therapeutic gene is limited toapproximately 4.9 kb, while longer sequences lead to truncation of AAVparticles. In some embodiments, the AAV vector is an AAV2/9 vector,e.g., AAV2 inverted terminal repeat (ITR) sequences cross-packaged intoAAV capsid.

In some embodiments, disclosed herein are cloning or expression vectorshaving, or having about, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 100%, or a range between any two of these values, sequenceidentity to one or more of the vectors disclosed herein. For example, insome embodiments, the cloning or expression vector comprises, orconsists of, a nucleotide sequence having at least about 85%, at leastabout 90%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99%, or more, sequence identity tothe full sequence of JBT-miR1 (SEQ ID NO: 85). In some embodiments, thevector comprises, or consists of, a nucleotide sequence having at leastabout 85%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or more,sequence identity to the nucleotide sequence of JBT-miR2. In someembodiments, the vector comprises, or consists of, a nucleotide sequencehaving about 85%, about 90%, about 95%, about 96%, about 97%, about 98%,about 99%, about 100%, or a range between any two of these values,sequence identity to the full sequence of JBT-miR1 (SEQ ID NO: 85) orJBT-miR2.

In some embodiments, the cloning vector or expression vector disclosedherein includes a nucleotide sequence having, or having about, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a range betweenany two of these values, sequence identity to each of the nucleotidesequences set forth in SEQ ID NOs: 59-64. In some embodiments, thecloning vector or expression vector disclosed herein includes anucleotide sequence having, or having about, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 100%, or a range between any two of thesevalues, sequence identity to each of the nucleotide sequences set forthin SEQ ID NOs: 86-89. In some embodiments, the cloning vector orexpression vector disclosed herein includes a nucleotide sequencehaving, or having about, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 100%, or a range between any two of these values, sequenceidentity to each of the nucleotide sequences set forth in SEQ ID NOs:59-64 and SEQ ID NOs: 86-89. In some embodiments, the cloning vector orexpression vector disclosed herein includes a nucleotide sequencehaving, or having about, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 100%, or a range between any two of these values, sequenceidentity to the nucleotide sequence of SEQ ID NO: 8

Therapeutic Compositions and Pharmaceutical Formulations

In another aspect, disclosed herein are embodiments of a therapeuticcomposition that includes an effective amount of at least onetherapeutic agent, and one or more of the followings: a) a compositioncomprising a plurality of microRNA (miR) antagonists as disclosedherein; b) an expression cassette as disclosed herein; and a cloning orexpression vector as disclosed herein.

While it is possible for the agents to be administered as the rawsubstances, it is preferable, in view of their potency, to present themas a pharmaceutical formulation. Thus, in some embodiments of thecompositions disclosed herein, the composition is further formulatedinto a pharmaceutical formulation. The term “pharmaceuticalformulation”, as used herein, refers to a composition suitable foradministering to an individual that includes a pharmaceutical agent. Forexample, a pharmaceutical formulation according to some aspects andembodiments of the present disclosure may comprise an anti-miRantagonist disclosed herein and a sterile aqueous solution. For example,the pharmaceutical formulations of the present disclosure for human usecomprise the agent, together with one or more acceptable carrierstherefor and optionally other therapeutic ingredients. The carrier(s)must be “acceptable” in the sense of being compatible with the otheringredients of the formulation and not deleterious to the recipientthereof or deleterious to the inhibitory function of the active agent.Desirably, the pharmaceutical formulations should not include oxidizingagents and other substances with which the agents are known to beincompatible.

Accordingly, some embodiments disclosed herein relate to pharmaceuticalformulations that include a therapeutic composition described herein anda pharmaceutically acceptable carrier. The formulations can alsocomprise additional ingredients such as diluents, stabilizers,excipients, and adjuvants. As used herein, “pharmaceutically acceptable”carriers, excipients, diluents, adjuvants, or stabilizers are the onesnontoxic to the cell or subject being exposed thereto (preferably inert)at the dosages and concentrations employed or that have an acceptablelevel of toxicity as determined by the skilled practitioner.

Buffers may also be included in the pharmaceutical formulations toprovide a suitable pH value for the formulation. Suitable such materialsinclude sodium phosphate and acetate. Sodium chloride or glycerin may beused to render a formulation isotonic with the blood. If desired, theformulation may be filled into the containers under an inert atmospheresuch as nitrogen or may contain an anti-oxidant, and are convenientlypresented in unit dose or multi-dose form, for example, in a sealedampoule.

The carriers, diluents and adjuvants can include antioxidants such asascorbic acid; low molecular weight polypeptides (e.g., less than about10 residues); proteins such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween™, Pluronics™ orpolyethylene glycol (PEG). In some embodiments, the physiologicallyacceptable carrier is an aqueous pH buffered solution.

Generally, the pharmaceutical formulations disclosed herein can beprepared by any one of the methods and techniques known in the art. Forexample, solid dosage forms can be prepared by wet granulation, drygranulation, direct compression, and the like. In some embodiments, thesolid dosage forms of the present disclosure may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. In some embodiments, the two components can beseparated by an enteric layer, which serves to resist disintegration inthe stomach and permit the inner component to pass intact into theduodenum or to be delayed in release. In these instances, a variety ofmaterials can be used for such enteric layers or coatings, suchmaterials including a number of polymeric acids and mixtures ofpolymeric acids with such materials as shellac, cetyl alcohol, andcellulose acetate.

Titers of the expression vector and/or one or more of the miRNAantagonists to be administered will vary depending, for example, on theparticular expression vector, the mode of administration, the treatmentgoal, the individual, and the cell type(s) being targeted, and can bedetermined by methods standard in the art.

As will be readily apparent to one of ordinary skill in the art, theuseful in vivo dosage of the expression vectors and/or one or more ofthe miRNA antagonists to be administered and the particular mode ofadministration will vary depending upon the age, weight, the severity ofthe affliction, and animal species treated, the particular expressionvector that is used, and the specific use for which the expressionvector and/or one or more of the miRNA antagonists is employed. Thedetermination of effective dosage levels, that is the dosage levelsnecessary to achieve the desired result, can be accomplished by one ofordinary skill in the art using routine pharmacological methods.Typically, human clinical applications of products are commenced atlower dosage levels, with dosage level being increased until the desiredeffect is achieved. Alternatively, acceptable in vitro studies can beused to establish useful doses and routes of administration of thecompositions identified by the present methods using establishedpharmacological methods.

For example, dosage regimens may be adjusted to provide the optimumdesired response. For example, a single dose may be administered, orseveral divided doses may be administered over time, or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions and formulations in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form, as usedherein, refers to physically discrete units suited as unitary dosagesfor the mammalian subjects to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe present disclosure are dictated by and directly dependent on (a) theunique characteristics of the therapeutic agent and the particulartherapeutic or prophylactic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

Thus, the skilled artisan would appreciate, based upon the disclosureprovided herein, that the dose and dosing regimen is adjusted inaccordance with methods well-known in the therapeutic arts. That is, themaximum tolerable dose can be readily established, and the effectiveamount providing a detectable therapeutic benefit to a patient may alsobe determined, as can the temporal requirements for administering eachagent to provide a detectable therapeutic benefit to the patient.Accordingly, while certain dose and administration regimens areexemplified herein, these examples in no way limit the dose andadministration regimen that may be provided to a patient in practicingthe present disclosure.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated, and may include single or multipledoses. It is to be further understood that for any particular subject,specific dosage regimens should be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition. Forexample, doses may be adjusted based on pharmacokinetic orpharmacodynamic parameters, which may include clinical effects such astoxic effects and/or laboratory values. Thus, the present disclosureencompasses intra-patient dose-escalation as determined by the skilledartisan. Determining appropriate dosages and regimens for administrationof therapeutic agents are well-known in the relevant art and would beunderstood to be encompassed by the skilled artisan once provided theteachings disclosed herein.

The expression vectors and/or the miRNA antagonists disclosed herein canbe administered to a subject (e.g., a human) in need thereof. The routeof the administration is not particularly limited. For example, atherapeutically effective amount of the recombinant viruses can beadministered to the subject by via routes standard in the art.Non-limiting examples of the route include intramuscular, intravaginal,intravenous, intraperitoneal, subcutaneous, epicutaneous, intradermal,rectal, intraocular, pulmonary, intracranial, intraosseous, oral,buccal, or nasal. In some embodiments, the recombinant virus isadministered to the subject by intramuscular injection. In someembodiments, the recombinant virus is administered to the subject byintravaginal injection. In some embodiments, the expression vectorsand/or the miRNA antagonists is administered to the subject by theparenteral route (e.g., by intravenous, intramuscular or subcutaneousinjection), by surface scarification or by inoculation into a bodycavity of the subject. In some embodiments, the expression vectorsand/or the miRNA antagonists are administered to muscle cells such as,cardiac muscle cells.

When administering these small miR oligonucleotide antagonists byinjection, the administration may be by continuous infusion, or bysingle or multiple boluses. The dosage of the administered miRantagonist will vary depending upon such factors as the patient's age,weight, sex, general medical condition, and previous medical history.Typically, it is desirable to provide the recipient with a dosage of themolecule which is in the range of from about 1 pg/kg to 10 mg/kg (amountof agent/body weight of patient), although a lower or higher dosage mayalso be administered,

In some embodiments, it may be desirable to target delivery of atherapeutic to the heart, while limiting delivery of the therapeutic toother organs. This may be accomplished by any one of a number of methodsknown in the art. In some embodiments, delivery to the heart of atherapeutic composition or pharmaceutical formulation described hereincomprises coronary artery infusion. In certain embodiments, coronaryartery infusion involves inserting a catheter through the femoral arteryand passing the catheter through the aorta to the beginning of thecoronary artery. In yet some other embodiments, targeted delivery of atherapeutic to the heart involves using antibody-protamine fusionproteins, such as those previously describe (Song E et al., NatureBiotechnology, 2005), to deliver the small miR oligonucleotideantagonists disclosed herein.

Actual administration of the expression vectors and/or the miRNAantagonists can be accomplished by using any physical method that willtransport the expression vectors and/or the miRNA antagonists into thetarget tissue of the subject. For example, the expression vectors and/orthe miRNA antagonists can be injected into muscle, the bloodstream,and/or directly into the liver. Pharmaceutical formulations can beprepared as injectable formulations or as topical formulations to bedelivered to the muscles by transdermal transport.

For intramuscular injection, solutions in an adjuvant such as sesame orpeanut oil or in aqueous propylene glycol can be employed, as well assterile aqueous solutions. Such aqueous solutions can be buffered, ifdesired, and the liquid diluent first rendered isotonic with saline orglucose. Solutions of the expression vectors and/or the miRNAantagonists as a free acid (DNA contains acidic phosphate groups) or apharmacologically acceptable salt can be prepared in water suitablymixed with a surfactant such as hydroxpropylcellulose. A dispersion ofthe expression vectors and/or the miRNA antagonists can also be preparedin glycerol, liquid polyethylene glycols and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The expression vectors and/or the miRNA antagonists to be used can beutilized in liquid or freeze-dried form (in combination with one or moresuitable preservatives and/or protective agents to protect the virusduring the freeze-drying process). For gene therapy (e.g., ofneurological disorders which may be ameliorated by a specific geneproduct) a therapeutically effective dose of the recombinant virusexpressing the therapeutic protein is administered to a host in need ofsuch treatment. The use of the expression vectors and/or the miRNAantagonists disclosed herein in the manufacture of a medicament forinducing immunity in, or providing gene therapy to, a host is within thescope of the present application.

In instances where human dosages for the expression vectors and/or themiRNA antagonists have been established for at least some condition,those same dosages, or dosages that are between about 0.1% and 500%,more preferably between about 25% and 250% of the established humandosage can be used. Where no human dosage is established, as will be thecase for newly-discovered pharmaceutical formulations, a suitable humandosage can be inferred from ED₅₀ or ID₅₀ values, or other appropriatevalues derived from in vitro or in vivo studies, as qualified bytoxicity studies and efficacy studies in animals

A therapeutically effective amount of the expression vectors and/or themiRNA antagonists can be administered to a subject at various points oftime. For example, the expression vectors and/or the miRNA antagonistscan be administered to the subject prior to, during, or after theinfection by a virus. The expression vectors and/or the miRNAantagonists can also be administered to the subject prior to, during, orafter the occurrence of a disease (e.g., cancer). In some embodiments,the expression vectors and/or the miRNA antagonists is administered tothe subject during cancer remission. In some embodiments, the expressionvectors and/or the miRNA antagonists is administered prior to infectionby the virus for immunoprophylaxis.

Alternatively or in addition, the dosing frequency of the expressionvectors and/or the miRNA antagonists can vary. For example, theexpression vectors and/or the miRNA antagonists can be administered tothe subject about once every week, about once every two weeks, aboutonce every month, about one every six months, about once every year,about once every two years, about once every three years, about onceevery four years, about once every five years, about once every sixyears, about once every seven years, about once every eight years, aboutonce every nine years, about once every ten years, or about once everyfifteen years. In some embodiments, the expression vectors and/or themiRNA antagonists is administered to the subject at most about onceevery week, at most about once every two weeks, at most about once everymonth, at most about one every six months, at most about once everyyear, at most about once every two years, at most about once every threeyears, at most about once every four years, at most about once everyfive years, at most about once every six years, at most about once everyseven years, at most about once every eight years, at most about onceevery nine years, at most about once every ten years, or at most aboutonce every fifteen years.

In some embodiments, a pharmaceutical kit is provided, wherein the kitcomprises: any of the forgoing the therapeutic compositions andpharmaceutical formulations, and written information (a) indicating thatthe formulation is useful for inhibiting, in myocardial cells, such as,for example cardiomyocytes, the function of a gene associated with theheart disease and/or (b) providing guidance on administration of thepharmaceutical formulation.

IV. Methods of the Disclosure

Some embodiments disclosed herein relate to a method for treating acardiac disease in a subject. The method includes administering orproviding to the subject a therapeutic composition suitable for thetreatment of cardiac diseases, wherein (a) the therapeutic compositionis a composition comprising a plurality of microRNA (miR) antagonists asdisclosed herein; (b) the therapeutic composition comprises anexpression cassette as disclosed herein; or (c) the therapeuticcomposition comprises a cloning or expression vector as disclosedherein.

Some embodiments of the disclosure relate to a method for promotingcardiac muscle regeneration in a subject. The method includesadministering or providing to the subject a therapeutic composition,wherein (a) the therapeutic composition is a composition comprising aplurality of microRNA (miR) antagonists as disclosed herein; (b) thetherapeutic composition comprises an expression cassette as disclosedherein; or (c) the therapeutic composition comprises a cloning orexpression vector as disclosed herein.

In some embodiments, a method for treating a cardiac disease orpromoting cardiac muscle regeneration in a subject as disclosed hereinoptionally includes a process of identifying or selecting the subject ashaving or suspected of having a cardiac disease. In some embodiments,the process of identifying or selecting is carried out prior toadministration of all therapeutic compositions and therapeutic agents ortherapies. In some embodiments, the process of identifying or selectingis carried out prior to administration of at least one of thetherapeutic composition and therapeutic agent or therapy.

In some embodiments, the cardiac disease is myocardial infarction,ischemic heart disease, heart failure (e.g., congestive heart failure),ischemic cardiomyopathy, hypertrophic cardiomyopathy, restrictivecardiomyopathy, alcoholic cardiomyopathy, viral cardiomyopathy,tachycardia-mediated cardiomyopathy, stress-induced cardiomyopathy,amyloid cardiomyopathy, arrhythmogenic right ventricular dysplasia, leftventricular noncompaction, endocardial fibroelastosis, aortic stenosis,aortic regurgitation, mitral stenosis, mitral regurgitation, mitralprolapse, pulmonary stenosis, pulmonary stenosis, pulmonaryregurgitation, tricuspid stenosis, tricuspid regurgitation, congenitaldisorder, genetic disorder, or a combination thereof. In some particularembodiments, the cardiac disease is myocardial infarction. In some otherparticular embodiments, the cardiac disease is Ischemic heart diseasewhere cardiac muscle regeneration is required. In yet some otherparticular embodiments, the cardiac disease is Duchenne musculardystrophy.

In another aspect, disclosed herein are embodiments of methods formodulating proliferation of a cardiomyocyte and/or muscle cell. Themethod includes (1) introducing into a cardiomyocyte a therapeuticcomposition, wherein (a) the therapeutic composition is a compositioncomprising a plurality of microRNA (miR) antagonists as disclosedherein; (b) the therapeutic composition comprises an expression cassetteas disclosed herein; or (c) the therapeutic composition comprises acloning or expression vector as disclosed herein; and (2) allowing thecardiomyocyte obtained from (1) to divide, thereby modulatingproliferation of the cardiomyocyte or muscle cell. In some embodiments,the introduction of the therapeutic composition into the cardiomyocyteincludes transfecting the cardiomyocyte and/or muscle cell with at leastone expression cassette or at least one viral vector comprising anucleic acid sequence encoding the plurality of miR antagonists. In someembodiments, the method further includes measuring the proliferation ofthe cardiomyocyte and/or muscle cell. In some embodiments, theproliferation of the cardiomyocyte and/or muscle cell is increasedcompared to a control cardiomyocyte and/or muscle cell lacking thenucleic acid sequence encoding the plurality of miR antagonists.

In some embodiments of the methods disclosed herein, the administrationstep can be performed on cells in cell-culture (i.e., ex-vivo) or oncells in a living body. Accordingly, in some embodiments, thecardiomyocyte and/or muscle cell is in vivo. In some other embodiments,the cardiomyocyte and/or muscle is ex vivo. In some embodiments, thecardiomyocyte and/or muscle is of a human subject. In some embodiments,the human subject is selected or identified as suffering from a cardiacdisease.

In some embodiments of the methods disclosed herein, where thetherapeutic composition or pharmaceutical formulation includesexpression cassettes or vectors comprising nucleotide sequences encodinga plurality of the miR antagonists as disclosed herein, the plurality ofmiR antagonists can be encoded by one or more expression cassettes orvectors. In some embodiments, the plurality of miR antagonists isencoded by a single expression cassette or vector. In some embodiments,the plurality of miR antagonists is encoded by 2, 3, 4, 5, 6, or moreexpression cassettes or vectors. In some embodiments, the plurality ofmiR antagonists can be encoded by the same type of expression cassetteor vector. In some embodiments, the plurality of miR antagonists can beencoded by different types of expression cassette or vector.

In some embodiments of the methods disclosed herein, where thetherapeutic composition or pharmaceutical formulation includes a cloningvector or expression vector, the vector can be derived from viruses,plasmids, cosmids, phages, or any combination thereof. In someembodiments, the vector is an integrating vector. In some embodiments,the vector is a viral vector. In some embodiments, the viral vector is alentiviral vector or an adeno-associated viral (AAV) vector. In someembodiments, the viral vector is an adeno-associated viral (AAV) vector.In some embodiments, the viral vector is an AAV2/9 vector.

In one aspect, disclosed herein are embodiments of methods forincreasing proliferation of a heart cell and/or increasing theexpression and/or activity of proteins involved in muscle structureand/or function and/or regeneration. The method includes contacting orproviding the heart cell with a combination of (1) a therapeuticcomposition, wherein (a) the therapeutic composition is a compositioncomprising a plurality of microRNA (miR) antagonists as disclosedherein; (b) the therapeutic composition comprises an expression cassetteas disclosed herein; or (c) the therapeutic composition comprises acloning or expression vector as disclosed herein; and (2) at least oneadditional therapeutic agent or therapy. In a related aspect, someembodiments disclosed herein relate to methods for inhibiting orreducing expression of a target microRNA (miR). The method includescontacting or providing the heart cell with a combination of (1) atherapeutic composition, wherein (a) the therapeutic composition is acomposition comprising a plurality of microRNA (miR) antagonists asdisclosed herein; (b) the therapeutic composition comprises anexpression cassette as disclosed herein; or (c) the therapeuticcomposition comprises a cloning or expression vector as disclosedherein; and (2) at least one additional therapeutic agent or therapy. Inthe methods according to the foregoing aspects, the heart cell cangenerally be any heart cell. Non-limiting examples of heart cellsuitable for the methods disclosed herein include cardiac fibroblasts,cardiac myocytes, endothelial cells, and vascular smooth muscle cells(VSMCs). In some embodiment, the heart cell is a cardiomyocyte or askeletal muscle cell. In some embodiments, the heart cell is acardiomyocyte. In some embodiments, the (miR) target gene is a geneassociated with a cardiac disease.

In yet another aspect, disclosed herein are embodiments of methods fortreating a muscular dystrophy (MD) disorder, comprising administering orproviding to the subject a therapeutic composition, wherein (a) thetherapeutic composition is a composition comprising a plurality ofmicroRNA (miR) antagonists as disclosed herein; (b) the therapeuticcomposition comprises an expression cassette as disclosed herein; or (c)the therapeutic composition comprises a cloning or expression vector asdisclosed herein, and wherein the administration of the therapeuticcomposition is performed in combination with an effective amount of atleast one additional therapeutic agent or at least one additionaltherapy to provide a combination therapy. In some embodiments, whereinthe muscular dystrophy disorder is associated with Amyotrophic LateralSclerosis (ALS), Charcot-Marie-Tooth Disease (CMT), Congenital MuscularDystrophy (CMD), Duchenne Muscular Dystrophy (DMD), Emery-DreifussMuscular Dystrophy (EDMD), Inherited and Endocrine Myopathies, MetabolicDiseases of Muscle, Mitochondrial Myopathies (MM), Myotonic MuscularDystrophy (MMD), Spinal-Bulbar Muscular Atrophy (SBMA), or a combinationthereof.

V. Combination Therapies

In some embodiments, the therapeutic compositions and pharmaceuticalformulations including the microRNA antagonists disclosed herein, suchas those provided in the Sequence Listing, or those including acombination of the microRNA antagonists disclosed herein, or anexpression cassette comprising a nucleotide sequence encoding one ormore microRNA antagonists disclosed herein, or a vector comprising oneor more of such expression cassettes, can be used in combination withone or more additional therapeutic agents. In some embodiments, thetherapeutic compositions and pharmaceutical formulations including themicroRNA antagonists disclosed herein, such as those provided in theSequence Listing, or those including a combination of the microRNAantagonists disclosed herein, or an expression cassette comprising anucleotide sequence encoding one or more microRNA antagonists disclosedherein, or a vector comprising one or more of such expression cassettes,can be used in combination with one or more therapeutic therapies.

Generally, any therapeutic approach pharmacological ornon-pharmacological for muscular dystrophies can be suitably employed asadditional therapeutic agents and therapies in the methods disclosedherein. Examples of additional therapeutic agents and therapies that canbe used in combination with the microRNA antagonists disclosed herein,or a composition or formulation that include a combination of themicroRNA antagonists disclosed herein, or an expression cassettecomprising a nucleotide sequence encoding one or more microRNAantagonists disclosed herein, or a vector comprising one or more of suchexpression cassettes, include, but are not limited to, Idebenone,Eplerenone, VECTTOR, AVI-4658, Ataluren/PTC124/Translarna,BMN044/PRO044, CAT-1004, any gene therapy for MD includingMicroDystrophin AAV gene therapy (SGT-001), Galectin-1 therapy (SB-002),LTBB4 (SB-001), rAAV2.5-CMV-minidystrophin, glutamine, NFKB inhibitors,sarcoglycan, delta (35 kDa dystrophin-associated glycoprotein), insulinlike growth factor-1 (IGF-1) expression, genome editing through theCRISPR/Cas9 system, any gene delivery therapy aimed at reintroducing afunctional recombinant version of the dystrophin gene, Exon skippingtherapeutics, read-through strategies for nonsense mutations, cell-basedtherapies, utrophin upregulation, myostatin inhibition,anti-inflammatories/anti-oxidants, mechanical support devices, anystandard therapy for muscular dystrophy, and combinations thereof.

Additional therapeutic agents useful for the methods of the presentdisclosure also include, but are not limited to, anti-platelet therapy,thrombolysis, primary angioplasty, Heparin, magnesium sulphate, Insulin,aspirin, cholesterol lowering drugs, angiotensin-receptor blockers(ARBs) and angiotensin-converting enzyme (ACE) inhibitors. Inparticular, ACE inhibitors have clear benefits when used to treatpatients with chronic heart failure and high-risk acute myocardialinfarction; this is possibly because they inhibit production ofinflammatory cytokines by angiotensin II. A non-limiting listing ofadditional therapeutic agents and therapies includes ACE inhibitors,such as Captopril, Enalapril, Lisinopril, or Quinapril; Angiotensin IIreceptor blockers, such as Valsartan; Beta-blockers, such as Carvedilol,Metoprolol, and bisoprolol; Vasodilators (via NO), such as Hydralazine,Isosorbide dinitrate, and Isosorbide mononitrate; Statins, such asSimvastatin, Atrovastatin, Fluvastatin, Lovastatin, Rosuvastatin orpravastatin; Anticoagulation drugs, such as Aspirin, Warfarin, orHeparin; or Inotropic agents, such as Dobutamine, Dopamine, Milrinone,Amrinone, Nitroprusside, Nitroglycerin, or nesiritide; CardiacGlycosides, such as Digoxin; Antiarrhythmic agents, such as Calciumchannel blockers, for example, Verapamil and Diltiazem or Class IIIantiarrhythmic agents, for example, Amiodarone, Sotalol or, defetilide;Diuretics, such as Loop diuretics, for example, Furosemide, Bumetanide,or Torsemide, Thiazide diuretics, for example, hydrochlorothiazide,Aldosterone antagonists, for example, Spironolactone or eplerenone.Alternatively or in addition, other treatments of cardiac disease arealso suitable, such as Pacemakers, Defibrillators, Mechanicalcirculatory support, such as Counterpulsation devices (intraaorticballoon pump or noninvasive counterpulsation), Cardiopulmonary assistdevices, or Left ventricular assist devices; Surgery, such as cardiactransplantation, heart-lung transplantation, or heart-kidneytransplantation; or immunosuppressive agents, such as Myocophnolatemofetil, Azathiorine, Cyclosporine, Sirolimus, Tacrolimus,Corticosteroids Antithymocyte globulin, for example, Thymoglobulin orATGAM, OKT3, IL-2 receptor antibodies, for example, Basilliximab orDaclizumab are also suitable.

In some embodiments, at least one of the additional therapeutic agentsor therapies includes a biologic drug. In some embodiments, the at leastone additional therapeutic agent or therapy comprises a gene therapy ortherapeutic gene modulation agent. As used herein, therapeutic genemodulation refers to the practice of altering the expression of a geneat one of various stages, with a view to alleviate some form of ailment.It differs from gene therapy in that gene modulation seeks to alter theexpression of an endogenous gene, for example through the introductionof a gene encoding a novel modulatory protein, whereas gene therapyconcerns the introduction of a gene whose product aids the recipientdirectly. Modulation of gene expression can be mediated at the level oftranscription by DNA-binding agents, which can be for example,artificial transcription factors, small molecules, or syntheticoligonucleotides. Alternatively or in addition, it can also be mediatedpost-transcriptionally through RNA interference.

The therapeutic compositions, pharmaceutical formulations disclosedherein and the additional therapeutic agents or therapies can be furtherformulated into final pharmaceutical preparations suitable for specificintended uses. In some embodiments, the therapeutic composition and theadditional therapeutic agent or therapy are administered in a singleformulation. In some embodiments, each of the therapeutic compositionand the additional therapeutic agent or therapy is administered in aseparate formulation. In some embodiments of the methods disclosedherein, the therapeutic composition and/or the additional therapeuticagent or therapy is administered to the subject in a single dose. Insome embodiments, the therapeutic composition and/or the additionaltherapeutic agent or therapy is administered to the subject in multipledosages. In some embodiments, the dosages are equal to one another. Insome embodiments, the dosages are different from one another. In someembodiments, the therapeutic composition and/or the additionaltherapeutic agent or therapy is administered to the subject in graduallyincreasing dosages over time. In some embodiments, the therapeuticcomposition and/or the additional therapeutic agent or therapy isadministered in gradually decreasing dosages over time.

The order of the administration of the therapeutic compositions andpharmaceutical formulations, with one or more additional therapeuticagent or therapy, can vary. In some embodiments, a therapeuticcomposition or pharmaceutical formulation disclosed herein can beadministered prior to the administration of all additional therapeuticagent or therapy. In some embodiments, a therapeutic composition orpharmaceutical formulation disclosed herein can be administered prior toat least one additional therapeutic agent or therapy. In someembodiment, a therapeutic composition or pharmaceutical formulationdisclosed herein can be administered concomitantly with one or moreadditional therapeutic agent or therapy. In yet still other embodiments,a therapeutic composition or pharmaceutical formulation disclosed hereincan be administered subsequent to the administration of at least oneadditional therapeutic agent or therapy. In some embodiments, atherapeutic composition or pharmaceutical formulation disclosed hereincan be administered subsequent to the administration of all additionaltherapeutic agent or therapy. In yet some embodiments, a therapeuticcomposition or pharmaceutical formulation disclosed herein and at leastone additional therapeutic agent or therapy are administered in rotation(e.g., cycling therapy). For examples, in some embodiments, atherapeutic composition or pharmaceutical formulation disclosed hereinand at least one additional therapeutic agent or therapy are cyclicallyadministered to a subject. Cycling therapy involves the administrationof a first active agent or therapy for a period of time, followed by theadministration of a second active agent or therapy for a period of timeand repeating this sequential administration. Cycling therapy can reducethe development of resistance to one or more therapies, avoid or reducethe side effects of one or more therapies, and/or improve the efficacyof treatment.

In some embodiments, intermittent therapy is an alternative tocontinuous therapy. For example, intermittent therapy can be used for aperiod of 6 months on, followed by a period of 6 months off. In someembodiments, one or more therapeutic agents or therapies are providedfor one month on, followed by one month off. In some embodiments, one ormore therapeutic agents or therapies are provided for three months on,followed by three months off. Accordingly, one or more of thetherapeutic compositions or pharmaceutical formulations disclosed hereincan be provided before, during and/or after administering one or moreadditional therapeutic agents or therapies, as described above.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

No admission is made that any reference cited herein constitutes priorart. The discussion of the references states what their authors assert,and the applicant reserves the right to challenge the accuracy andpertinence of the cited documents. It will be clearly understood that,although a number of information sources, including scientific journalarticles, patent documents, and textbooks, are referred to herein, thisreference does not constitute an admission that any of these documentsforms part of the common general knowledge in the art.

The discussion of the general methods given herein is intended forillustrative purposes only. Other alternative methods and alternativeswill be apparent to those of skill in the art upon review of thisdisclosure, and are to be included within the spirit and purview of thisapplication.

EXAMPLES

Additional embodiments are disclosed in further detail in the followingexamples, which are not in any way intended to limit the scope of thisdisclosure or the claims.

Example 1 Design of Inhibitory Oligonucleotides for Specific microRNAs

This Example demonstrates the design and composition of syntheticoligonucleotides that can be used as antagonists of miR-99a-5p,miR-100-5p, Let-7a-5p, and Let-7c-5p.

Nucleotide sequences of the following human microRNAs were analyzed:miR-99a-5p, miR-100-5p, Let-7a-5p and Let-7c-5p. The sequences of thesemicroRNAs and the sequences of the complementary antagonists are shownin Table 5 below. The bases highlighted in bold font correspond to basedifferences between let-7a-5p and let-7c-5p, or between miR-99a-5p andmiR-100-5p. The seed sequence of all microRNAs is generally consideredto be bases 2-8 starting from the 5′ end. Without wishing to be bound byany particular theory, the nucleobases within the seed sequence of amicroRNA are believed to be the bases that make the biggest contributionto deciding which mRNAs will be targeted by the microRNA. In thesequences listed in Table 5 below, the seed sequences are underlined.

TABLE 5 Nucleotide sequences of human miR-99-5p,miR-100-5p, Let-7a-5p, and Let-7c-5p andthe complementary inhibitory sequencesthat can be incorporated into any suitablevectors such as, for example, viral vectorfor cardiac muscle generation. >hsa-let-7a-5p MIMAT0000062 5′-UGA GGU AGU AGG UUG UAU AGUU-3′ (SEQ ID NO: 1)  Sense3′-ACU CCA UCA UCC AAC AUA UCAA-5′ (SEQ ID NO: 2) Anti-sense >hsa-let-7c-5p MIMAT0000064 5′-UGA GGU AGU AGG UUG UAU GGUU-3′ (SEQ ID NO: 3)  Sense3′-ACU CCA UCA UCC AAC AUA CCAA-5′ (SEQ ID NO: 4) Anti-sense >hsa-miR-99a-5p MIMAT0000097 5′-AAC CCG UAG AUC CGA UCU UGUG-3′ (SEQ ID NO: 5)  Sense3′-UUG GGC AUC UAG GCU AGA ACAC-5 (SEQ ID NO: 6) Anti-sense >hsa-miR-100-5p MIMAT0000098 5′-AAC CCG UAG AUC CGA ACU UGUG-3′ (SEQ ID NO: 7)  Sense3′-UUG GGC AUC UAG GCU UGA ACAC-5′ (SEQ ID NO: 8)  Anti-sense

To further assess the sequence conservation of the correspondingmicroRNA homologs from different mammalian species were also examined.As shown in Table 6 below, the nucleotide sequences of miR-99a-5p,miR-100-5p, Let-7a-5p and Let-7c-5p from different mammalian specieswere observed to exhibit high degrees of sequence homology. Thenucleotide sequences of Let-7a-5p are 100% homologous across all speciesanalyzed. The nucleotide sequences of Let-7c-5p are also 100% homologousacross all species analyzed. The sequence of miR-99a-5p from dog lacksnucleobase #, otherwise all other sequences are homologous. Dog ismissing miR-100 miRNA, otherwise all other sequences are homologous.

TABLE 6 Sequence homology of miR-99a-5p, miR-100-5p, Let-7a-5p andLet-7c-5p homologs. Dre: Danin rerio (zebrafish), Hsa: Homosapiens (human), Ptr: Pan troglodytes (chimpanzee), Cfa:Canis familiaris (dog), Ssc: Sus scrofa (minipig), Rno:Rattus norvegicus (rat), Mmu: Mus musculus (mouse). dre-let-7a-5pUGAGGUAGUAGGUUGUAUAGUU (SEQ ID NO: 9) mmu-let-7a-5pUGAGGUAGUAGGUUGUAUAGUU (SEQ ID NO: 10) rno-let-7a-5pUGAGGUAGUAGGUUGUAUAGUU (SEQ ID NO: 11) ssc-let-7a-5pUGAGGUAGUAGGUUGUAUAGUU (SEQ ID NO: 12) ptr-let-7a-5pUGAGGUAGUAGGUUGUAUAGUU (SEQ ID NO: 13) hsa-let-7a-5pUGAGGUAGUAGGUUGUAUAGUU (SEQ ID NO: 14) cfa-let-7a-5pUGAGGUAGUAGGUUGUAUAGUU (SEQ ID NO: 15) dre-let-7c-5pUGAGGUAGUAGGUUGUAUGGUU (SEQ ID NO: 16) mmu-let-7c-5pUGAGGUAGUAGGUUGUAUGGUU (SEQ ID NO: 17) rno-let-7c-5pUGAGGUAGUAGGUUGUAUGGUU (SEQ ID NO: 18) ssc-let-7c-5pUGAGGUAGUAGGUUGUAUGGUU (SEQ ID NO: 19) ptr-let-7c-5pUGAGGUAGUAGGUUGUAUGGUU (SEQ ID NO: 20) hsa-let-7c-5pUGAGGUAGUAGGUUGUAUGGUU (SEQ ID NO: 21) cfa-let-7c-5pUGAGGUAGUAGGUUGUAUGGUU (SEQ ID NO: 22) dre-miR-99a-5pAACCCGUAGAUCCGAUCUUGUG 22 (SEQ ID NO: 23) mmu-miR-99a-5pAACCCGUAGAUCCGAUCUUGUG 22 (SEQ ID NO: 24) rno-miR-99a-5pAACCCGUAGAUCCGAUCUUGUG 22 (SEQ ID NO: 25) cfa-miR-99aAACCCGUAGAUCCGAUCUUGU 21 (SEQ ID NO: 26) ssc-miR-99aAACCCGUAGAUCCGAUCUUGUG 22 (SEQ ID NO: 27) ptr-miR-99aAACCCGUAGAUCCGAUCUUGUG 22 (SEQ ID NO: 28) hsa-miR-99a-5pAACCCGUAGAUCCGAUCUUGUG 22 (SEQ ID NO: 29) dre-miR-100-5pAACCCGUAGAUCCGAACUUGUG (SEQ ID NO: 30) mmu-miR-100-5pAACCCGUAGAUCCGAACUUGUG (SEQ ID NO: 31) rno-miR-100-5pAACCCGUAGAUCCGAACUUGUG (SEQ ID NO: 32) ssc-miR-100AACCCGUAGAUCCGAACUUGUG (SEQ ID NO: 33) ptr-miR-100AACCCGUAGAUCCGAACUUGUG (SEQ ID NO: 34) hsa-miR-100-5pAACCCGUAGAUCCGAACUUGUG (SEQ ID NO: 35)

A total of twenty (20) anti-miR oligonucleotide compounds were designed,including ten for the let-7a-5p/let-7c-5p family and ten for themiR-99a-5p/miR-100-5p family. Two anti-miR designs targeting Let-7c-5pare JRX0100, JRX0102 and could be used to inhibit Let-7a-5p. Twoanti-miR designs targeting Let-7a-5p are JRX0101 and JRX0103 and couldbe used to inhibit Let-7a-5p. Six anti-miR designs targeting bothlet-7a-5p and Let-7c-5p are JRX0104, JRX0105, JRX0106, JRX0107, JRX0108,and JRX0109. Five anti-miR designs targeting miR-100a are JRX0110,JRX0113, JRX0115, JRX0117, and JRX0119. Five anti-miR designs targetingmiR-99a are JRX0111, JRX0112, JRX0114, JRX0116, and JRX0118. In thisexperiment, the designs used locked nucleic acid (LNA) chemistrymodifications (+), in which the 2′-O-oxygen is bridged to the 4′position via a methylene linker to form a rigid bicycle, locked into aC3′-endo (RNA) sugar conformation allowing for resistance to nucleasedegradation and extremely high affinity for its complementary RNA base.These modifications were particularly incorporated at each end of themolecules as designated by (+) in the sequences in TABLE 7 forstability, by e.g. enhancing resistance to exonucleases, and in theregion complementary to the seed to increase affinity for their targetedmiR and thus increased potency as a microRNA inhibitor. The backbone ofthe anti-miRs is phosphorothioate (indicated by * in Table 7 below) toenable a broad distribution in animals. This type of backbone functionsby steric blockade of a specific microRNA in the RISC complex. Theanti-miR oligonucleotide compounds were carefully kept relatively short,to avoid the possible of forming heteroduplexes, but long enough to bindplasma proteins efficiently and keep them from being filtered out ofcirculation in the kidneys and thus improve their biodistributionproperties. A summary of 20 anti-miR designs and their respective targetmicroRNAs is shown in Table 7 below.

As indicated in Table 7, some of the miR-7 family anti-miRs are 100%homologous to both let-7c-5p and c isoforms of interest and will inhibitboth members. In contrast, the miR-99a-5p and miR-100 family anti-miRsare each only 100% homologous to one of the family members due to theposition of the one base that is different in these miRs. However, inreality all of the anti-miRs designed for each of the two families caninhibit both members of the family of interest because, similarly totarget recognition, the seed region (bases 2-8) is the most importantregion for determining anti-miR activity.

TABLE 7 Summary of twenty anti-miR designs disclosed herein No. No.Nomenclature/ SEQ LNAs Stretch LNAs in Nomenclature/Sequence/ Sequence/ID Name Target Length PLUS of DNA Seed * Structure Structure NO JRX0100let-7c 19 9 3 5 +C*+C*A*T*+A*C*A*A*+C*C*T CCATACAACCTA 36*A*+C*T*+A*C*+C*+T*+C CTACCTC JRX0101 let-7a 19 9 3 5+C*+T*A*T*A*+C*A*A*C*+C*T CTATACAACCTA 37 *A*+C*+T*A*C*+C*+T*+C CTACCTCJRX0102 let-7c 18 9 3 5 +C*+A*T*A*C*A*+A*C*C*T*A* CATACAACCTAC 38+C*T*+A*+C*C*+T*+C TACCTC JRX0l03 let-7a 18 9 3 5+T*+A*T*A*C*+A*A*C*+C*T*A TATACAACCTAC 39 *C*+T*+A*+C*C*+T*+C TACCTCJRX0104 let-7a/c 17 9 3 5 +A*T*A*C*A*+A*C*C*+T*A*+C ATACAACCTACT 40*T*+A*+C*C*+T*+C ACCTC JRX0105 let-7a/c 17 9 3 5+A*+T*A*C*A*+A*C*C*T*A*+C ATACAACCTACT 41 *T*+A*C*+C*+T*+C ACCTC JRX0106let-7a/c 16 8 3 5 +T*+A*C*A*A*+C*C*T*A*+C*T TACAACCTACTA 42*+A*+C*C*+T*+C CCTC JRX0107 let-7a/c 16 8 3 5 +T*+A*C*A*A*+C*C*T*A*+C*TTACAACCTACTA 43 *+A*C*+C*+T*+C CCTC JRX0108 let-7a/c 15 8 3 5+A*+C*A*A*C*+C*T*A*+C*T*A ACAACCTACTAC 44 *+C*C*+T*+C CTC JRX0109let-7a/c 15 9 3 6 +A*+C*A*A*+C*C*T*A*+C*+T* ACAACCTACTAC 45+A*C*+C*+T*+C CTC JRX0l10 miR-100 19 9 3 5 +C*+A*A*G*+T*T*C*G*+G*A*TCAAGTTCGGATC 46 *C*+T*A*+C*G*+G*+G*+T TACGGGT JRX0111 miR-99 19 9 3 5+C*+A*A*G*A*+T*C*G*G*+A*T CAAGATCGGATC 47 *C*+T*+A*C*G*+G*+G*+T TACGGGTJRX0l12 miR-99 18 9 3 5 +A*+A*G*+A*T*C*G*+G*A*T*C AAGATCGGATCT 48*+T*A*+C*+G*G*+G*+T ACGGGT JRX0l13 miR-100 18 9 3 5+A*+A*G*T*T*+C*G*G*+A*T*C AAGTTCGGATCT 49 *T*+A*+C*+G*G*+G*+T ACGGGTJRX0l14 miR-99 17 9 3 5 +A*+G*A*T*C*+G*G*A*+T*C*+ AGATCGGATCTA 50T*A*+C*+G*G*+G*+T CGGGT JRX0l15 miR-100 17 9 3 5+A*+G*T*T*C*+G*G*+A*T*C*+ AGTTCGGATCTA 51 T*A*+C*G*+G*+G*+T CGGGTJRX0l16 miR-99 16 8 3 5 +G*+A*T*C*G*+G*A*T*C*+T*A GATCGGATCTAC 52*+C*+G*G*+G*+T GGGT JRX0l17 miR-100 16 8 3 5 +G*+T*T*C*G*+G*A*T*C*+T*AGTTCGGATCTAC 53 *+C*G*+G*+G*+T GGGT JRX0l18 miR-99 15 8 3 5+A*+T*C*G*G*+A*T*C+T*+A*+ ATCGGATCTACG 54 C*+G*G*+G*+T GGT JRX0l19miR-100 15 9 3 6 +T*+T*C*G*+G*A*T*C*+T*+A* TTCGGATCTACG 55 +C*G*+G*+G*+TGGT

As described in further detail below, the inhibitory activity of thesesynthetic anti-miRs can be subsequently assessed by using a commerciallyreporter vector system, pMIR-REPORT™ miRNA Expression Reporter VectorSystem, made available by Applied Biosystems® (see, e.g., FIG. 3) (PartNumber AM5795, Applied Biosystems). In this system, microRNA bindingsites of interest are inserted the multiple cloning sites locateddownstream of the coding sequence of the reporter luciferase.

Example 2 Design of Adeno-Viral Vector JBT-miR1

This Example summarizes experimental results illustrating the design ofa modified hairpin Zip construct and vector expressing inhibitorysequences of the microRNAs miR-99a, miR-100-5p, miR-Let-7a-5p, andmiR-Let-7c-5p using RNAi technology. In this experiment, RNAi technologywas implemented within a target cell in the form of a base-pair shorthairpin (sh) RNA (shRNA), which is processed into an approximately 20base pair small interfering RNA through the endogenous miR pathway. Asmall hairpin RNA or short hairpin RNA (shRNA) is typically defined asan artificial RNA molecule with a tight hairpin turn that can be used tosilence target gene expression via RNA interference (RNAi). To evaluatethe potential therapeutic use of anti-miR-99/100 and anti-Let-7a/c toregenerate cardiac muscle in the murine heart, two recombinant virusesexpressing complementary inhibitory sequences to Let-7a/c and miR-99/100were made by AAV2 Inverted Terminal Repeat (ITR) sequences crosspackaged into AAV9 capsids (AAV2/9). The AAV2/9 serotype has clearcardiac tropism. Viral delivery of complementary sequences to miRs is acommon approach. In this experiment, AAV vectors were selected as beingoptimal in cardiovascular gene therapy since they a) contain no viralprotein-coding sequences to stimulate an immune response, b) do notrequire active cell division for expression to occur and c) have asignificant advantage over adenovirus vectors because of their stable,long-term expression of recombinant genes in cardiomyocytes in vivo.

In this experiment, a modified hairpin Zip construct expressing (1) theLet-7a-5p and miR-99a-5p inhibitory sequences under the H1 promoter andU6 promoter, respectively; and (2) Let-7c-5p and miR-100-5p inhibitorysequences under the regulation of the H1 promoter and U6 promoter,respectively. A summary of the nucleotide sequences of anti-miRantagonists and loop sequence inserted into the pAV-4in1shRNA-GFP vectorto generate the viral vector JBT-miR1 is provided in Table 8 below. Inthis experiment, the nucleotide sequences encoding the foregoingantagonists were cloned in the pAV-4in1shRNA-GFP vector (FIG. 1). Thenucleotide sequences corresponding to the four miR inhibitory sequenceswere inserted into the pAV-4in1shRNA-GFP vector between the ITR sites ofthe vector and specifically within the BamH1 and HindIII cloning site,and were separated by a loop sequence, TGTGCTT (SEQ ID NO: 56). In theresulting vector, expression of each inhibitory sequence was regulatedby alternate human U6 promoter or the H1 promoter driving the expressionof a short hairpin RNA (shRNA) against miR-99a-5p, 100, Let-7a-5p andLet-7c.

As shown in FIG. 1, also inserted into the vector was a CMV promoterdriving the expression of a Green Fluorescent Protein (GFP) reporter,which in turn allows for detection in various tissues for preclinicalstudies, followed by a Simian virus 40 (SV40) sequence which is apolyomavirus binding site that initiates DNA replication at the originof replication allowing for replication of in mammalian cells expressingSV40 large T. It is contemplated however that, these sequences can alsobe suitably removed from vectors designed for use in human drugs.

Vector genomes with AAV2 ITR sequences were cross-packaged into AAV9capsids via triple transfection of AAV-293 cells (J. Fraser Wright,Human Gene Therapy, 20:698-706, July 2009), and then purified byiodixanol gradient centrifugation. Titers of the AAV vectors, which isdefined as viral genomes (vg)/ml, were then determined by a qPCR-basedassay. In this experiments, the following primers were used foramplifying the mouse U6 promoter: 5′-TCGCACAGACTTGTGGGAGAA-3′ (SEQ IDNO: 57) (forward) and 5′ CGCACATTAAGCCTCTATAGTTACTAGG-3′ (SEQ ID NO: 58)(reverse).

Known copy numbers of plasmids carrying the corresponding expressioncassettes were used to construct standard curves for quantification. Thevirus was manufactured and sequenced by Vigene Biosciences Inc.(Rockville, Md.) using manufacturer's recommended safety precautions andprocedures.

TABLE 8 Summary of the nucleotide sequences ofanti-miR antagonists and loop sequenceinserted into the BamH1 and HindIIIcloning site of the pAV-4in1shRNA-GFPvector to generate the viral vector JBT-miR1. SEQ ID Target Hairpin NOlet-7a-5p GTGAGGTAGTAGGTTGTATAGTTT 59 CAAGAGAACTATACAACCTACTAC CTCATTTTTmiR-99a-5p GAACCCGTAGATCCGATCTTGTGT 60 CAAGAGCACAAGATCGGATCTACGGGTTTTTTT (H1-)let-7a-  GTGAGGTAGTAGGTTGTATAGTTT 61 5p & (U6)- CAAGAGAACTATACAACCTACTAC miR-99a-5p CTCATTTTTGAGCTCAAAAAAACCCGTAGATCCGATCTTGTGCTCTTG ACACAAGATCGGATCTACGGGTTC let-7c-5pGTGAGGTAGTAGGTTGTATGGTTT 62 CAAGAGAACCATACAACCTACTAC CTCATTTTTmiR-100-5p GAACCCGTAGATCCGAACTTGTGT 63 CAAGAGCACAAGTTCGGATCTACGGGTTTTTTT (H1-)let-7C- GTGAGGTAGTAGGTTGTATGGTTT 64 5p & (U6)-CAAGAGAACCATACAACCTACTAC miR-100-5p CTCATTTTTGAGCTCAAAAAAACCCGTAGATCCGAACTTGTGCTCTTG ACACAAGTTCGGATCTACGGGTTC

The nucleotide sequence of the JBT-miR1 viral vector design is set forthat SEQ ID NO: 85 in the Sequence Listing.

As described in Example 1 above, a total of twenty (20) anti-miRoligonucleotide compounds were designed. The sequences of these anti-miRoligonucleotide compounds are shown in Table 9 below. Any combination ofthe sequences of anti-miR oligonucleotide compounds disclosed in Table 9below can be inserted into the BamH1 and HindIII cloning site of thepAV-4in1shRNA-GFP vector to generate other viral delivery systems formiR-99a, miR-100-5p, Let-7a-5p and Let-7c-5p inhibition.

TABLE 9 No. No. LNAs Stretch LNAs in SEQ ID Target Length PLUS of DNASeed * Sequence NO let-7c 19 9 3 5 CCATACAACCTACTACCTC 65 let-7a 19 9 35 CTATACAACCTACTACCTC 66 let-7c 18 9 3 5 CATACAACCTACTACCTC 67 let-7a 189 3 5 TATACAACCTACTACCTC 68 let-7a/c 17 9 3 5 ATACAACCTACTACCTC 69let-7a/c 17 9 3 5 ATACAACCTACTACCTC 70 let-7a/c 16 8 3 5TACAACCTACTACCTC 71 let-7a/c 16 8 3 5 TACAACCTACTACCTC 72 let-7a/c 15 83 5 ACAACCTACTACCTC 73 let-7a/c 15 9 3 6 ACAACCTACTACCTC 74 miR-100 19 93 5 CAAGTTCGGATCTACGGGT 75 miR-99 19 9 3 5 CAAGATCGGATCTACGGGT 76 miR-9918 9 3 5 AAGATCGGATCTACGGGT 77 miR-100 18 9 3 5 AAGTTCGGATCTACGGGT 78miR-99 17 9 3 5 AGATCGGATCTACGGGT 79 miR-100 17 9 3 5 AGTTCGGATCTACGGGT80 miR-99 16 8 3 5 GATCGGATCTACGGGT 81 miR-100 16 8 3 5 GTTCGGATCTACGGGT82 miR-99 15 8 3 5 ATCGGATCTACGGGT 83 miR-100 15 9 3 6 TTCGGATCTACGGGT84

The nucleotide sequence of the JBT-miR1 viral vector design is set forthat SEQ ID NO: 85 in the Sequence Listing.

Example 3 Inhibitory Activity of Viral Vector JBT-miR1 in Myocardium InVivo

This Example summarizes experimental results demonstrating that theviral vector JBT-miR1 constructed as described in Example 2 can decreaselate gadolinium enhancement of the LV in CD1 mice.

In this experiment, CD1 mice were anesthetized with Ketamine (100 mg/kg)and Xylazine (10 mg/kg) and intubated with a pressure ventilator (KentScientific, CT). Throughout the procedure, the animal was intubated viathe trachea, and mechanically ventilated with room air (respiratory rate55-65 breaths/min, tidal volume 2.5 ml) (Model 687-Harvard Apparatus). Askin incision was made from the midsternal line toward the left armpit,and the chest opened with a 1-cm lateral cut along the left side of thesternum, cutting between the 3rd and 4th ribs to expose the LV. Theascending aorta and main pulmonary artery would be then identified andthe LAD located between the left and right ventricles (RV). LADocclusion was performed by tying an 8-0 PROLENE® suture ligature on apiece of PE-10 tubing. Blanching of the territory of perfusion of theLAD, along with acute ST segment elevation on limb-lead EKG leads, and awhitening of the LV would certify vessel occlusion.

JBT-miR1 or control virus was then administered at a dose of 6×1011vg/mouse diluted in 60 μl of saline by intracardiac injection into themyocardium bordering the infarct zone using an insulin syringe withincorporated 30-gauge needle. The mice were left for 3 weeks and thensubject to cardiac MRI. As shown in Table 10, it was observed that micetransformed with JBT-miR1 were found to decrease late gadoliniumenhancement of the LV in CD1 mice with permanent LAD ligation 3 weeksfollowing an intracardiac injection of JBT-miR1 compared with a virusexpressing GFP.

TABLE 10 LV LGE MI Mass Size (% ID BW (g) AAV (mg) LV mass) GFP Control10 36 GFP Control 162.5 10.68 11 36 GFP Control 165.9 27.59 Mean 36164.2 19.14 SD 0 2.4 11.96 JBT 13 39 JBT 204.0 14.31 14 36 JBT 156.810.90 Mean 38 180.4 12.61 SD 2 33.4 2.41

FIGS. 2A-2B pictorially summarize the results of cardiac MRI imagingexperiments in which the cardiac MRI images of control GFP virus (FIG.2A) versus JBT-miR1 (FIG. 2B) were demonstrated to decrease lategadolinium enhancement of the LV in CD1 mice with permanent LAD ligation3 weeks following an intracardiac injection of vector JBT-miR1 whencompared with a virus expressing GFP. For this model male, CD1 mice(8-12 weeks of age) weighing ˜30-40 grams were subject to permanentischemia as before. In this experiment, MRI was performed on ahorizontal Bruker Biospec® 7T/20 MRI system (Bruker, Germany) onanesthetized mice (SA Instruments, NY). For end-diastolic (ED) andend-systolic (ES) images, volumetric data were determined from theproduct of compartment area and slice thickness (1 mm). LVED and ESvolumes (EDV and ESV) were calculated from the summation of all slicesand the EF derived. EDV multiplied by myocardial specific gravity, y(1.055 g/cm3) calculates LV mass. MI size: ED images of each slice wereselected for scar delineation. The sizes of the contrast-enhanced areasin the MR images were plotted against the corresponding areas obtainedfrom TTC staining. Infarction size was expressed as the % of LV mass.

As illustrated in Table 11, two-dimensional (2D) echocardiographyanalysis showed a significant increase in cardiac output of CD1 micewith permanent LAD ligation 3 weeks following an intracardiac injectionof JBT-miR1 compared with a virus expressing GFP. In this experiment,2D-Echo was performed on anesthetized mice on Day 5 and Day 14 and day21 by using a Hewlett-Packard/Phillips 5500 machine and a 15-MHztransducer.

TABLE 11 Cardiac Output (μL/min) ID Type 5 Day 2 Week 3 Week 10 AAV-GFP11537.2 7951.7 12984.0 11 AAV-GFP 16287.7 9265.4 15015.0 12 AAV-GFP10833.1 13410.9 10848.0 Mean 12886.0 10209.3 12949.0 SD 2966.9 2849.42083.7 13 AAV-JBT-miR1 18893.2 22919.6 23414.6 14 AAV-JBT-miR1 17134.013918.5 22494.0 16 AAV-JBT-miR1 13492.2 14359.3 18612.0 17 AAV-JBT-miR15670.0 8409.5 21129.6 Mean 13797.4 14901.7 21412.6 SD 5866.5 5991.62089.7 Ttest GFP vs. 0.8001 0.2346 0.0044 JBT

Example 4 Design of Viral Vector JBT-miR2

This Example summarizes experimental results illustrating the design ofanother viral vector, named JBT-miR2, which expresses tough decoys (alsoknown as TuDs) that can be superior to zips (JBT-miR1) (Takeshi et al.2009). In brief, four 120-based oligonucleotide sequences were insertedinto between the ITR sites of the vector and in the BamH1 and HindIIIcloning site to generate the TuDs that can inhibit the let-7 andmiR-99a-5p families when inserted into a viral delivery system. In thenucleotide sequences of the foregoing oligonucleotides shown below, boldcharacters correspond to the respective miR binding sites.

let-7a-5p  (SEQ ID NO: 86)GACGGCGCTAGGATCATCAACAACTATACAACCAATGTACTACCTCACAAGTATTCTGGTCACAGAATACAACAACTATACAACCAATGTACTACCTCAC AAGATGATCCTAGCGCCGTC.let-7a-5p Reverse Complement  (SEQ ID NO: 87)GACGGCGCTAGGATCATCTTGTGAGGTAGTACATTGGTTGTATAGTTGTTGTATTCTGTGACCAGAATACTTGTGAGGTAGTACATTGGTTGTATAGTTG TTGATGATCCTAGCGCCGTCmiR-99a-5p  (SEQ ID NO: 88)GACGGCGCTAGGATCATCAACCACAAGATCGGAAATGTCTACGGGTACAAGTATTCTGGTCACAGAATACAACCACAAGATCGGAAATGTCTACGGGTAC AAGATGATCCTAGCGCCGTCmiR-99a-5p Reverse Complement  (SEQ ID NO: 89)GACGGCGCTAGGATCATCTTGTACCCGTAGACATTTCCGATCTTGTGGTTGTATTCTGTGACCAGAATACTTGTACCCGTAGACATTTCCGATCTTGTGG TTGATGATCCTAGCGCCGTC.

In some experiments, restriction sites were added to theoligonucleotides which in turn facilitate their subcloning into theappropriate vectors. The 5′ end of these sequences were cloned adjacentto the promoter sequence (e.g., the U6 promoter) and the 3′ end wascloned against a PolII termination sequence (e.g., TTTTT).

Example 5 In Vitro Bioactivity of Anti-miR Oligonucleotides

This Example summarizes the results of the experiments performed toassess the activity of MicroRNA (miR) antagonists (anti-miRs) tolet-7a/c and miR-99/100, ex vivo in rat neonatal ventricular myocytesand Hela cells, using the pMIR-REPORT™ miRNA Expression Reporter VectorSystem (Part Number AM5795, Applied Biosystems®). The pMIR-REPORT™ miRNAExpression Reporter Vector System consists of an experimental fireflyluciferase reporter vector and an associated β-gal reporter controlplasmid. By inserting predicted miRNA target sequences in the multiplecloning site located downstream of the coding sequence of the reporter,these vectors are often used to conduct accurate, quantitativeevaluations of miRNA function. This system is also often used toevaluate siRNA target sites and to analyze the influence of 3′ UTRsequences on gene expression.

Without being bound be any particular theory, it is believed that theunmodified pMIR-REPORT™ should have maximal luciferase activity whentransfected in to Hela cells or rat neonatal ventricular cardiacmyocytes. Stated differently, by inserting the predicted miRNA targetsequences for miR-99 (Luciferase reporter 1, LUC 1), miR-100 (Luciferasereporter 2, LUC 2) and Let-7a-5p (Luciferase reporter 3, LUC 3) andLet-7c-5p (Luciferase reporter 4, LUC 4) into the multiple cloning siteof the pMIR-REPORT™, the luciferase activity of the resulting vectors(LUC 1, LUC2, LUC3 and LUC 4) would be significantly less than thepMIR-REPORT™ alone.

The modified Luciferase miRNA Expression Reporter Vectors constructed asdescribed above, i.e., pMIR-REPORT LUC 1, LUC 2, LUC 3 and LUC 4; can beused to conduct accurate, quantitative evaluations of miRNA function,such that inhibition of endogenous miR members in HeLa cells and cardiacmyocytes would lead to a dose-dependent increase in luciferase activitycompared to the LUC 1, LUC 2, LUC 3 and LUC 4 vectors alone.

Method

Complementary sequences to the microRNAs miR-99, miR-100, Let-7a-5p andLet-7c-5p were designed and cloned into the multiple cloning site of thepMIR-REPORT™ miRNA Expression Reporter Vector System. The resultingvectors were named LUC 1, LUC 2, LUC 3 and LUC 4 expression vectors,respectively. Hela cells were cultured in 96 well tissue culture platesand co-transfected with 50 ng/well of purified DNA of a modified LUCvector (i.e., LUC 1, LUC 2, LUC 3, or LUC 4 vector) and 10 ng/well of aBeta-Galactosidase (β-gal) reporter plasmid, together with increasingconcentrations of anti-miRs (0-50 nM) for up to 5 hours usingLipofectamine® 2000 Reagent (Life Technologies). Similarly, neonatal ratventricular cardiac myocytes were cultured in 24 well tissue cultureplates and co-transfected with 500 ng/well of LUC 1, LUC 2, LUC 3, andLUC 4 DNA and 100 ng/well of β-gal reporter plasmid to confirmtransfection efficiency. At forty-eight hours after transfection, thetransfected cells were harvested and the cell lysates were assayed forLuciferase activity and β-gal activity. The luciferase activity wasnormalized to β-gal activity and expressed as fold activation over theLUC 1, LUC 2, LUC 3, and LUC 4 plasmids alone.

FIG. 3 schematically shows the pMIR-REPORT™ Luciferase miRNA expressionreporter vector, which contains a firefly luciferase reporter gene underthe control of a cauliflower virus (CMV) promoter/termination system.The 3′ UTR of the luciferase gene contains a multiple cloning site forinsertion of predicted miRNA binding targets or other nucleotidesequences. By inserting a predicted miRNA target sequence into themultiple cloning sites of the pMIR-REPORT vector, the luciferasereporter can be then subjected to regulation that mimics the miRNAtarget.

FIG. 4 schematically shows the pMIR-REPORT™ miRNA β-Galactosidaseexpression reporter vector carrying the reporter gene β-Galactosidase,which is designed for transfection normalization. Typically, β-galexpression from this control plasmid can be used to normalizevariability due to differences in cell viability and transfectionefficiency.

Construction of the pMIR-REPORT™ vectors for miR-99, miR-100, Let-7a-5pand let-7c:

The following oligonucleotides (in bold) were purchased from IntegratedDNA Technologies (IDT), San Diego. Underlined are the miRNA bindingsites corresponding to the sequences complementary with miRNA, whichwere subsequently inserted downstream of the coding sequence of theluciferase gene.

>hsa-miR-99a-5p MIMAT0000097 (SEQ ID NO: 90) 5′-AAC CCG UAG AUC CGA UCU UGUG-3′ (SEQ ID NO: 91) CACA AGA TCG GAT CTA CGG GTT 99a FORWARD PRIMER:  (SEQ ID NO: 92) 5′-AACACTAGT CACAAGATCGGATCTACGGGTT AAGCTTGTT-3′ 99a REVERSE PRIMER: (SEQ ID NO: 93) 5′-AACAAGCTTAACCCGUAGAUCCGAUCUUGUGACTAGTGTT-3′ >hsa-miR-100-5p MIMAT0000098 (SEQ ID NO: 94)  5′-AAC CCG UAG AUC CGA ACU UGUG-3′ (SEQ ID NO: 95) CACA AGT TCG GAT CTA CGG GTT 100 FORWARD PRIMER:  (SEQ ID NO: 96) 5′-AACACTAGT CACAAGTTCGGATCTACGGGTT AAGCTTGTT-3′ 100 REVERSE PRIMER: (SEQ ID NO: 97) 5′-AACAAGCTTAACCCGUAGAUCCGAACUUGUGACTAGTGTT-3′ >hsa-let-7a-5p MIMAT0000062 (SEQ ID NO: 98)  5′-UGA GGU AGU AGG UUG UAU AGUU-3′ (SEQ ID NO: 99) AACT ATA CAA CCT ACT ACC TCA LET7A FORWARD PRIMER:  (SEQ ID NO: 100) 5′-AACACTAGT AACTATACAACCTACTACCTCA AAGCTTGTT-3′ LET7A REVERSE PRIMER: (SEQ ID NO: 101) 5′-AACAAGCTTUGAGGUAGUAGGUUGUAUAGUUACTAGTGTT-3′ >hsa-let-7c-5p MIMAT0000064 (SEQ ID NO: 102)  5′-UGA GGU AGU AGG UUG UAU GGUU-3′ (SEQ ID NO: 103) AACC ATA CAA CCT ACT ACC TCA LET7C FORWARD PRIMER:  (SEQ ID NO: 104) 5′-AACACTAGT AACCATACAACCTACTACCTCA AAGCTTGTT-3′ LET7C REVERSE PRIMER: (SEQ ID NO: 105)  5′-AACAAGCTTUGAGGUAGUAGGUUGUAUGGUUACTAGTGTT-3′

The vector contains the following ordered elements:5′-luciferase-Multiple Cloning Site (MCS) allowing for insertingnucleotide sequences corresponding to desired miRNA binding sites intoits 3′UTR. The MCS contains the following ordered restriction sites:5′-SpeI-HindIII. The SpeI (ACTAGT) and HindIII (AAGCTT) were selected asthe restriction enzymes because they both function well in the samebuffer (NEB2). The oligonucleotides contains the following orderedelements: 5′-AAC-SpeI site-(miRNA binding site)-HindIII site-GTT-3′. Thenucleotides AAC (and GTT) are extra nucleotides allowing restrictionenzymes to bind more effectively.

The nucleotide sequences for the pMIR-REPORT™ Luciferase vectors formiR-99, miR-100, Let-7a-5p and Let-7c-5p (LUC1, LUC2, LUC3, and LUC4respectively) are set forth in SEQ ID NOs: 106-109 of the SequenceListing.

HELA TRANSFECTION: Hela cells were cultured in Minimum Essential Mediawith Earle's Balanced Salt Solution (HyClone™) supplemented with 2 mML-glutamine, 1 mM sodium pyruvate, 1 nM Non-essential Amino Acids, and10% FBS (PAA) and penicillin streptomycin. The cells were plated inserum-containing media without antibiotics in 96-well plates (1×10⁴cells/well) 24 hours prior to transfection and were at a confluency ofbetween 30-70% at the time of transfection.

Cells were then transfected with 50 ng/well of the LUC reporter plasmidand 10 ng/well of the β-gal reporter plasmid for 2 hours with 0.1, 1, 10or 50 nanomol/L (nM) using Lipofectamine 2000 (Life Technologies, Cat#11668-019) according to the manufacturer's instructions using Opti-MEM®Medium and normal growth medium in a final volume of 200 μl/well.Reporter plasmids (pMIR-REPORT™ or LUC plasmid) were transfected alone.

A typical plate setup for Hela cells in 2×96 wells is shown below, wherecolumn 6 of each plate identifies the LUC vector used for transfection.

TABLE 12 Plate 1 with Luciferase Reporter 1 miR-99 (LUC 1) andLuciferase Reporter 2 miR-100 (LUC2) JRX0- 1 2 3 4 5 6 7 8 9 10 11 120.1 n A 111 112 114 116 118 LUC 1 111 112 114 116 118 Lipo  1 B 111 112114 116 118 LUC 1 111 112 114 116 118 Lipo 10 C 111 112 114 116 118 LUC1 111 112 114 116 118 Lipo 50 D 111 112 114 116 118 LUC 1 111 112 114116 118 Lipo 0.1 nM E 110 113 115 117 119 LUC 2 110 113 115 117 119 pMIR 1 F 110 113 115 117 119 LUC 2 110 113 115 117 119 pMIR 10 G 110 113 115117 119 LUC 2 110 113 115 117 119 pMIR 50 H 110 113 115 117 119 LUC 2110 113 115 117 119 pMIR

TABLE 13 Plate 2 with Luciferase Reporter 3 Let-7a-5p (LUC 3) andLuciferase Reporter 4 Let-7c-5p (LUC4) JRX0- 1 2 3 4 5 6 7 8 9 10 11 120.1 nM A 101 103 104 105 106 LUC 3 107 108 109 101 103 Lipo (10 nM) (10nM) (10 nM) (10 nM) (10 nM) (10 nM) 1 B 101 103 104 105 106 LUC 3 107108 109 101 103 Lipo (10 nM) (10 nM) (10 nM) (10 nM) (10 nM) (10 nM) 10C 101 103 104 105 106 LUC 3 107 108 109 101 103 Lipo (50 nM) (50 nM) (50nM) (50 nM) (50 nM) (50 nM) 5 D 101 103 104 105 106 LUC 3 107 108 109101 103 Lipo (50 nM) (50 nM) (50 nM) (50 nM) (50 nM) (50 nM) 0.1 nM E100 102 104 105 106 LUC 4 107 108 109 100 102 pMIR (10 nM) (10 nM) (10nM) (10 nM) (10 nM) (10 nM) 1 F 100 102 104 105 106 LUC 4 107 108 109100 102 pMIR (10 nM) (10 nM) (10 nM) (10 nM) (10 nM) (10 nM) 10 G 100102 104 105 106 LUC 4 107 108 109 100 102 pMIR (50 nM) (50 nM) (50 nM)(50 nM) (50 nM) (50 nM) 50 H 100 102 104 105 106 LUC 4 107 108 109 100102 pMIR (50 nM) (50 nM) (50 nM) (50 nM) (50 nM) (50 nM)

CARDIAC MYOCYTE TRANSFECTION: neonatal rat cardiomyocytes were isolatedand plated on Primaria™ coated plates at density of 80,000 cells perwell (24 well). Twenty-four hours after plating the cells weretransfected with 500 ng/well of the LUC reporter plasmid and 100 ng/wellof the β-gal reporter plasmid for 5 hours with 0.1, 1, 3, 10 or 50nanomol/L (nM) using Lipofectamine® 2000 (Life Technologies, Cat#11668-019) according to the manufacturer's instructions using Opti-MEM®Medium and normal growth medium in a final volume of 600 μl/well.Reporter plasmids (pMIR-REPORT™ or LUC plasmid) were transfected alone.

A typical plate setup for cardiac myocytes was as follows:

Plate 1 Luciferase Reporter 1 (LUC1) miR-99

0 nM LUC 1 LUC 1 114 (3 nM) 116 (3 nM) LUC 1 (3 nM) Lipo 0.1 nM JRX0111JRX0112 JRX0114 JRX0116 JRX0118 Lipo 10 nM JRX0111 JRX0112 JRX0114JRX0116 JRX0118 pMIR 50 nM JRX0111 JRX0112 JRX0114 JRX0116 JRX0118 pMIRPlate 2 Luciferase Reporter 2 (LUC 2) miR-100

0 nM LUC 2 LUC 2 JRX0115 (3 nM) JRX0117 (3 nM) JRX0119 (3 nM) Lipo 0.1nM JRX0110 JRX0113 JRX0115 JRX0117 JRX0119 Lipo 10 nM JRX0110 JRX0113JRX0115 JRX0117 JRX0119 pMIR 50 nM JRX0110 JRX0113 JRX0115 JRX0117JRX0119 pMIRPlate 3 Luciferase Reporter 3 (LUC 3) let-7a

0 nM LUC 3 LUC 3 104 (3 nm) 106 (3 nM) 108 (3 nM) Lipo 0.1 nM JRX0101103 104 (10 nm) 106 (10 nm) 108 (10 nM) Lipo 10 nM JRX0101 103 105 (3nm) 107 (3 nM) 109 (3 nM) pMIR 50 nM JRX0101 103 105 (10 nm) 107 (10 nM)109 (10 nM) pMIRPlate 4 Luciferase Reporter 4 (LUC 4) let07c

0 nM LUC 4 LUC 4 JRX0104 (3 nm) JRX0106 (3 nM) JRX0108 (3 nM) Lipo 0.1nM JRX0100 JRX0102 JRX0104 (10 nm) JRX0106 (10 nm) JRX0108 (10 nM) Lipo10 nM JRX0100 JRX0102 JRX0105 (3 nm) JRX0107 (3 nM) JRX0109 (3 nM) pMIR50 nM JRX0100 JRX0102 JRX0105 (10 nm) JRX0107 (10 nM) JRX0109 (10 nM)pMIR

The above experiments were repeated.

Promoter Activity Assay

Cells were grown at 37° C. and harvested 48 hours post transfection forluciferase and β-gal assays in normal growth media using ONE-Glo™ Luc(Promega™ # E6110), Beta-Glo® Luc (Promega™ # E4720) and Glo LysisBuffer (Promega™ # E2661). Luciferase activity was measured using theBioTek Synergy™ HT. Promoter activity was expressed as Fold over theLuciferase 1, 2, 3 or 4 plasmid alone and was normalized to β-galactivity levels.

Statistical Analysis

Luciferase activity was normalized to β-gal and the data were expressedas fold activation of the respective LUC vector alone. The fold data forthe Hela experiments represent a single experiment. The experiments wererepeated to confirm the results. The fold date from the two separatecardiac myocyte experiments were combined since the cells were culturedand transfected on the same day. The data are presented at Mean StandardDeviation. Graphs were drawn using GraphPad Prism 7 software with thenormalized fold increase in luciferase activity (x-axis) against thelog-10 M concentration of anti-miR (y-axis).

Results

A test experiment on the Luciferase constructs was conducted to confirmthat Luciferase Construct 1 (LUC 1, miR-99), Luciferase Construct 2 (LUC2, miR-100), Luciferase Construct 3 (LUC 3, let-7a), and LuciferaseConstruct 4 (LUC 4, let-7c), had significantly less luciferase activitycompared to the unmodified pMIR-REPORT Vector, suggesting that theendogenous miRs (Let-7a-5p miR-99a, miR-100-5p, miR-Let-7c5p,miR-Let-7a-5p) within Hela cells can bind to the respective LUCconstruct and repress luciferase activity. In this experiment, Helacells were transfected with each of the LUC constructs and then treatedwith the corresponding anti-miRs. It was contemplated that the anti-miRswould compete with their corresponding endogenous microRNA in Helacells. The unmodified pMIR-REPORT™ were observed to provide maximalluciferase activity when transfected in to Hela cells or rat neonatalventricular cardiac myocytes. By inserting the predicted miRNA targetsequences in the multiple cloning site for miR-99 (Luciferase reporter1, LUC 1), miR-100 (Luciferase reporter 2, LUC 2) and Let-7a-5p(Luciferase reporter 3, LUC 3) and Let-7c-5p (Luciferase reporter 4, LUC4), luciferase activity was significantly less than the pMIR-REPORT™alone. The modified pMIR-REPORT LUC 1, LUC 2, LUC 3 and LUC 4 LuciferasemiRNA Expression Reporter Vectors can be used to conduct accurate,quantitative evaluations of miRNA function, such that inhibition ofendogenous miR members in HeLa cells and cardiac myocytes would lead toa dose-dependent increase in luciferase activity compared to the LUC 1,LUC 2, LUC 3 and LUC 4 vectors alone. Without being bound to anyparticular theory, it was believed anti-miRs act via steric blockade ofa specific microRNA in the RISC complex and increase the correspondingLuciferase promoter activity. The result of this test experiment isschematically summarized in FIG. 5.

In a subsequent experiment performed with Hela cells, it was observedthat JRX0111, JRX0112, JRX0114, JRX0116, JRX0118 miR-99a anti-miRsincreased Luciferase Construct 1 (LUC 1, miR-99) activity in adose-dependent manner (FIG. 6).

In a similar manner, as shown in FIG. 7, JRX0110, JRX0113, JRX0115,JRX0117, JRX0119 miR-100-5p anti-miRs were observed to increaseLuciferase Construct 2 (LUC 2, miR-100) activity in Hela cells in adose-dependent manner.

Similarly, JRX0101, JRX0103, JRX0104, JRX0105, JRX0106, JRX0107,JRX0108, JRX0109 Let-7a-5p miR-Let-7a-5p anti-miRs were also found toincrease Luciferase Construct 3 (LUC 3, let-7a) activity in Hela cellsin a dose-dependent manner (FIGS. 8A-8B); and JRX0100, JRX0102, JRX0104,JRX0105, JRX0106, JRX0107, JRX0108, JRX0109 Let-7c-5p miR-Let-7c5panti-miRs were observed to increase Luciferase Construct 4 (LUC 4,let-7c) activity in Hela cells in a dose-dependent manner (FIGS. 9A-9B).

In various experiments performed with neonatal rat ventricular cardiacmyocytes, JRX0111, JRX0112, JRX0114, JRX0116, JRX0118 miR-99a anti-miRswere observed to increase Luciferase Construct 1 (LUC 1, miR-99)activity in a dose-dependent manner (FIG. 10); JRX0110, JRX0113,JRX0115, JRX0117, JRX0119 miR-100-5p anti-miRs were observed to increaseLuciferase Construct 2 (LUC 2, miR-100) activity in a dose-dependentmanner (FIG. 11); JRX0101, JRX0103, JRX0104, JRX0105, JRX0106, JRX0107,JRX0108, JRX0109 Let-7a-5p miR-Let-7a-5p anti-miRs were observed toincrease Luciferase Construct 3 (LUC 3, let-7a) activity in adose-dependent manner (FIGS. 12A-12B) and JRX0100, JRX0102, JRX0104,JRX0105, JRX0106, JRX0107, JRX0108, JRX0109 Let-7c-5p miR-Let-7c5panti-miRs were also found to increase Luciferase Construct 4 (LUC 4,let-7c) activity in a dose-dependent manner (FIGS. 13A-13B).

CONCLUSION

Taken together, the experimental data presented above confirm thepotency, specificity and activity of the specified anti-miRs. Themodified plasmids LUC 1, LUC 2, LUC 3 and LUC 4 were found to exhibitsignificantly less luciferase activity compared to the pMIR-REPORT™empty plasmid. It was further observed that each antagonist from thecorresponding miR family dose dependently activated their respective LUCreporter plasmid. In addition, it appears that the anti-miRs designed toinhibit the following microRNAs miR-99, miR-100, Let-7a-5p, andLet-7c-5p bound to their specific target mRNA with varying efficiency inboth cell types tested.

All of the references disclosed herein, including but not limited tojournal articles, textbooks, patents and patent applications, are herebyincorporated by reference for the subject matter discussed herein and intheir entireties. Throughout this disclosure, various informationsources are referred to and incorporated by reference. The informationsources include, for example, scientific journal articles, patentdocuments, textbooks, and World Wide Web browser-inactive pageaddresses. The reference to such information sources is solely for thepurpose of providing an indication of the general state of the art atthe time of filing. While the contents and teachings of each and everyone of the information sources can be relied on and used by one of skillin the art to make and use the embodiments disclosed herein, anydiscussion and comment in a specific information source should no way beconsidered as an admission that such comment was widely accepted as thegeneral opinion in the field.

What is claimed is:
 1. A method for promoting cardiac muscleregeneration for treating a cardiac disease in a subject, comprisingadministrating to a subject a plurality of microRNA (miR) antagonists,wherein said plurality of miR antagonists comprises one or moremiR-99a-5p antagonists, one or more miR-100-5p antagonists, one or moremiR-Let-7a-5p antagonists, and one or more miR-Let-7c-5p antagonists,wherein one or more of the followings applies: a. at least one of theone or more miR-99a-5p antagonists comprises an anti-miR-99a-5pcomprising a chemically modified nucleotide sequence selected from thegroup consisting of JRX0111, JRX0112, JRX0114, JRX0116 and JRX0118; b.at least one of the one or more miR-100-5p antagonists comprises ananti-miR-100-5p comprising a chemically modified nucleotide sequenceselected from the group consisting of JRX0110, JRX0113, JRX0115, JRX0117and JRX0119; c. at least one of the one or more Let-7a-5p antagonistscomprises an anti-miR-Let-7a-5p comprising a chemically modifiednucleotide sequence selected from the group consisting of JRX0101,JRX0103, JRX0104, JRX0105, JRX0106, JRX0107, JRX0108, and JRX0109; andd. at least one of the one or more Let-7c-5p antagonists comprises ananti-miR-Let-7c-5p comprising a chemically modified sequence selectedfrom the group consisting of JRX0100, JRX0102, JRX0104, JRX0105,JRX0106, JRX0107, JRX0108, and JRX0109.
 2. The method of claim 1,wherein at least one of the anti-miRs comprises one or more chemicalmodifications selected from the group consisting of a modifiedinternucleoside linkage, a modified nucleotide, and a modified sugarmoiety, and combinations thereof, optionally wherein at least one of theone or more chemical modifications comprises a modified nucleotide,optionally wherein the modified nucleotide comprises a locked nucleicacid (LNA) chemistry modification, a peptide nucleic acid (PNA), anarabino-nucleic acid (FANA), an analogue, a derivative, or a combinationthereof, and optionally wherein the locked nucleic acid (LNA) isincorporated at one or both ends of the modified anti-miR.
 3. The methodof claim 2, wherein at least one of the one or more chemicalmodifications comprises a modified internucleoside linkage, optionallywherein the modified internucleoside linkage is selected from the groupconsisting of a phosphorothioate, 2′-Omethoxyethyl (MOE), 2′-fluoro,alkylphosphonate, phosphorodithioate, alkylphosphonothioate,phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate,carboxymethyl ester, and combinations thereof, and optionally whereinthe modified internucleoside linkage comprises a phosphorothioateinternucleoside linkage
 4. The method of claim 2, wherein at least oneof the one or more chemical modifications comprises a modified sugarmoiety.
 5. The method of claim 4, wherein the modified sugar moiety is a2′-O-methoxyethyl modified sugar moiety, a 2′-methoxy modified sugarmoiety, a 2′-O-alkyl modified sugar moiety, a bicyclic sugar moiety, ora combination thereof, optionally wherein the modified sugar moietycomprises a 2′-O-methyl sugar moiety.
 6. The method of claim 1, whereinadministering to the subject a plurality of miR antagonists comprisesadministering to the subject a pharmaceutical composition comprising theplurality of miRNA antagonists.
 7. The method of claim 1, furthercomprising administering to the subject at least one therapeutic agent,wherein the at least one therapeutic agent is selected from the groupconsisting of Idebenone, Eplerenone, VECTTOR, AVI-4658,Ataluren/PTC124/Translarna, BMN044/PRO044, CAT-1004, MicroDystrophin AAVGene Therapy (SGT-001), Galectin-1 Therapy (SB-002), LTBB4 (SB-001),rAAV2.5-CMV-minidystrophin, Glutamine, NFKB inhibitors, Sarcoglycan,delta (35 kDa dystrophin-associated glycoprotein), insulin like growthfactor-1 (IGF-1), and combinations thereof.
 8. The method of claim 7,wherein the plurality of miR antagonists and the at least onetherapeutic agent are administered to the subject sequentially.
 9. Themethod of claim 7, wherein the plurality of miR antagonists and the atleast one therapeutic agent are administered to the subject in rotation.10. The method of claim 1, wherein the cardiac disease is myocardialinfarction, ischemic heart disease, dilated cardiomyopathy, heartfailure (e.g., congestive heart failure), ischemic cardiomyopathy,hypertrophic cardiomyopathy, restrictive cardiomyopathy, alcoholiccardiomyopathy, viral cardiomyopathy, tachycardia-mediatedcardiomyopathy, stress-induced cardiomyopathy, amyloid cardiomyopathy,arrhythmogenic right ventricular dysplasia, left ventricularnoncompaction, endocardial fibroelastosis, aortic stenosis, aorticregurgitation, mitral stenosis, mitral regurgitation, mitral prolapse,pulmonary stenosis, pulmonary regurgitation, tricuspid stenosis,tricuspid regurgitation, congenital disorder, genetic disorder, or acombination thereof.
 11. The method of claim 1, comprising modulatingproliferation of a cardiomyocyte and/or muscle cell in the subject. 12.The method of claim 1, comprising increasing proliferation of a heartcell and/or increasing the expression and/or activity of proteinsinvolved in muscle structure and/or function and/or regeneration in thesubject.
 13. The method of claim 1, comprising inhibiting or reducingexpression of a target microRNA (miR) in the subject.
 14. The method ofclaim 1, wherein the subject is a human subject.
 15. The method of claim14, wherein the human subject suffers from a cardiac disease.
 16. Themethod of claim 14, wherein the human subject suffers from a musculardystrophy (MD) disorder.
 17. The method of claim 1, wherein theplurality of miR antagonists is expressed from one or more expressionvectors.
 18. The method of claim 17, wherein the plurality of miRantagonists is expressed from a single expression vector.
 19. The methodof claim 18, wherein the single expression vector is a viral vector. 20.The method of claim 19, wherein viral vector is an adeno-associatedviral (AAV) vector.